3rd Edition

An Introduction to Antibodies
and Their Applications

EMD Millipore is a division of

Merck KGaA, Darmstadt, Germany
By:
Manpreet Mutneja, Ph.D.
Chandra Mohan, Ph.D.
Kevin D. Long, Ph.D.
Chandreyee Das, Ph.D.

In collaboration with:
John L. Hermesman
Robin T. Clark, Ph.D.
Robert Brockett, HTL (ASCP)CM

Acknowledgements:
Mary Ann Ford, Marketing
Communications, and
Liza Benson, Design for their
contributions and dedication
to make this guide possible.

EMD Millipore
An Introduction to Antibodies
and Their Applications
EMD Millipore – your partner in Life Science research.
At EMD Millipore, our commitment to advancing scientific research defines us.
We understand the challenges faced by today’s researcher and know the importance of research tools (antibodies,
proteins, enzymes, inhibitors, and other reagents) for research, drug discovery, and publications. This technical
guide on the theory and practical use of antibodies in biological research is a part of our continuing commitment
to provide useful information and exceptional service to researchers.

The 3rd edition of An Introduction to Antibodies and Their Applications provides a concise overview of some
of the key features for the use of antibodies and immunochemical techniques in biological research. This handy
reference guide supplements the techniques described in literature, recorded in general laboratory procedures,
and described on individual product data sheets. Antibody design, development, and production are our expertise.
Stringent validation of our antibodies is only one component of a comprehensive process we undertake to provide
the antibodies most cited by the research community (see section Antibody Quality on page 2 for an in-depth look
at our expertise).

As every antibody and experimental design is unique, these general principles and suggestions should not be
interpreted as applicable to all situations, but rather as an additional source of information. As always, individual
assays must be optimized empirically and antibody titers must be established for every unique batch of antibody.

Whether you are a veteran researcher or just beginning your research career, we hope that you will find this guide
to be useful in your research. Your suggestions and comments for further improvements are always welcome.

EMD Millipore products are among the best in the industry, and include the expertise of Chemicon®, Upstate®,
Calbiochem®, and Novagen®. For more information about using any of our research products, including more than
10,000 antibodies and kits, or simply to get scientific advice for a variety of immunological applications, please
contact us. In addition, you can find extensive general information and technical specifications on our website
www.emdmillipore.com. Rest assured that our highly trained, exceptional customer and technical service support
specialists are always available to support you in your research.

1
A Note on Antibody Quality
It is often assumed that because specificity defines certain, uncommon immunization protocols are robust
their function, antibodies must have some intrinsic enough to use the less immunogenic, but more specific
high quality, which implies reliability and ease of use. peptides critical for generating a quality antibody.
Surprisingly, this is most often not the case. Although
it is true that antibody binding to a specific amino acid Based on the combined strengths of Chemicon®,
sequence or conformational form can be theoretically Upstate®, Millipore®, Calbiochem® and Novagen®,
modeled and is predictable in theory, decades of at EMD Millipore, we have decades of experience in
user experience has demonstrated that factors such innovative immunogen design, immunization, selection,
as antigenicity and uniqueness of the immunogen screening, and validation to create many of our highly
sequence, antibody concentration, buffers, immunization cited antibodies. Our commitment to produce high
and sample preparation techniques, contribute quality antibodies is based not only on innovation, but is
significantly to issues of cross-reactivity and nonspecific also tempered with customer beta testing and feedback
binding. to the design/engineering team, prior to release. These
efforts and collaborations have lead to the development
Since the early 1970s, when researchers began to use of new validation techniques and novel antibody-based
antibodies as protein probes, the problem of inconsistent technologies, such as improved multiplexing and high
antibody performance has plagued researchers. The resolution imaging flow cytometry. This cycle, from
pioneer developers and users of antibodies had fairly thoughtful design to field usage and report, is the basis
extensive knowledge of immunology to rely on. In today’s of quality. Our technical and scientific background
research world, antibodies are used for an amazing is what makes EMD Millipore a major designer and
variety of applications, from purification to blocking producer of quality antibodies, and not merely a
of cellular function. Today, antibody users range from distributor. Indeed, there is a whole lot of science in
classic medical anatomists to novice biochemists and every vial of our antibodies.
biomedical engineers. Many users are unaware that
immunogen design is fairly complex and that only

Advanced Collaboration,
immunization Multi-platform discovery &
technologies validation publication

R
1

C H
H C
N N
H O

Innovative Rigorous Customer

immunogen selection experimentation
design & purification

2
The science inside your tube of Special validation
EMD Millipore antibodies. To support our multi-step, multi-application validation
process, we have a tissue and blot library with over
1300 lysates, allowing us to precisely determine each
Antigen development and immunization
antibody’s specificity. At EMD Millipore, we have the
Our team of antibody research scientists continually
advantage of having an entire cell analysis technology
monitors and reviews the latest publications and
development team in-house. We validate antibodies for
collaborates with leading research institutions to identify
flow cytometry using our own guava easyCyte™ dual-
the most useful targets and antibodies. We design
laser microcapillary instruments. Similarly, our in-house
multiple immunogens, taking into consideration post-
bead-based immunodetection team helps us validate
translational modifications, structure, cross-reactivity,
antibodies using the trusted Luminex xMAP platform. Our
and homology. We discuss target selection with leaders
microarray system handles development and validation
in key research areas, including neuroscience, cancer,
mainly for antibodies recognizing modified histones.
epigenetics, and signaling research using their expertise
Using confocal microscopes and high-throughput IHC
to guide development and validation.
instruments, we can obtain accurate data faster than
manual imaging. Further scientific review determines
Monoclonal antibody development
whether staining patterns conform to published
Following immunization at our USDA-approved facility,
subcellular expression. For immunohistochemistry, we
we develop monoclonal and polyclonal antibodies using
include negative controls, to confirm the signal.
state-of-the-art processes. For monoclonal antibodies,
we use a robotic cloning and screening system, which
handles all the fusions and feeding steps for hybridomas.
Technical review
The final step in our process is a quality review carried
These highly automated processes use cutting-edge
out by an independent team of scientists. Before
technology to ensure optimum throughput and
releasing the antibody to manufacturing, the team
maximum consistency. Using an array-jet automated
considers all antibody validation data, as well as data
microarray system, we test the fusion and identify
from scientists who participated in the beta testing. If
positive clones. To ensure that the positive signal is from
our antibodies fail to meet our strictest specifications,
the antibody, we screen again using ELISA. Positives are
they are discarded without question, even if they have hit
screened using automated Western blotting of cells with
initial targets. As an integral component of our quality
endogenously expressed antigens. The pool of positives is
process, only antibodies that pass this stringent review
repeatedly diluted to isolate the highest performer until
are made available to customers.
our samples reach 100% clonality. This additional process
is one way that EMD Millipore sets itself apart from the
Customer experimentation
competition. With additional Western blotting, we select
and collaboration
the best possible clone for further validation.
Once produced and released for sale, we support
customers’ research efforts with a highly specialized
Polyclonal antibody development team of technical support scientists and field engineers.
Polyclonal antibodies are purified using fast protein We work closely with researchers to improve immunogen
liquid chromatography (FPLC) systems, using no column design and antibody performance. We have a growing
more than 10 times. Automated Western blotting is network of beta testers to continue validation. And each
used to determine further purification steps. These new and every one of our antibodies is backed by our 100%
polyclonal antibodies are then validated in a number Antibody Performance Guarantee.
of applications and are also incorporated into kits and
assays for EMD Millipore’s protein and cellular analysis EMD Millipore antibodies are among the most cited,
platforms. trusted, and highly validated on the market today.
Their quality starts at inception and carries through
manufacturing, production and distribution - into the lab
and onto the bench. From start to finish, it’s the science
in every tube of EMD Millipore antibodies that assures
confidence in the world’s most reliable, defensible, and
publishable antibody performance.
3
 This is an interactive PDF document with clickable links.
Jump to the sections of your choice from either the main or chapter section listings.
Click on catalogue numbers and other resources to go directly to the web for additional information.

Click on at the bottom of any page to return to the main Table of Contents

4
Table of Contents
Foreword
A Note on Antibody Quality

1 Antibody Theory page 7

1.1 An Introduction to Antibodies 7
1.2 Antigens 7
1.3 Epitopes 8
1.4 Antibodies 9
1.5 Antibody-Antigen Interaction 12
1.6 Nature of Antigen-Antibody Bonds 13
1.7 Factors Affecting Antigen-Antibody Reactions 13
1.8 Generation of Antibodies 14
1.9 Antibody Formats 16
1.10 Biological Effects of Antibodies 17

2 Antibody Practice page 21

2.1 Selection and Use 21
2.2 Antibody Titer and Concentration 22
2.3 Storage and Handling of Antibodies 22
2.4 Conjugated Antibodies 23
2.5 Use of Secondary Antibodies 24
2.6 Proper Controls 25
2.7 Publishing with Antibodies 26
2.8 Validating Antibodies 26

3 Antibody Applications page 29

3.1 Introduction to Antibody Applications 29
3.2 Immunoprecipitation 30
3.3 Chromatin Immunoprecipitation (ChIP) 34
3.4 Western Blotting 42
3.5 Enzyme-linked Immunosorbent Assays (ELISA) 51
3.6 Multiplexed Bead-based Detection 56
3.7 Immunohistochemistry/Immunocytochemistry 60
3.8 Flow Cytometry 68
3.9 Functional Blocking and Stimulation Assays 76

4 Sample Protocols page 81

4.1 Immunoprecipitation 81
4.2 Chromatin Immunoprecipitation (ChIP) 81
4.3 Western Blotting 83
4.4 Enzyme-linked Immunosorbent Assays (ELISA) 85
4.5 Multiplexed Bead-based Detection 86
4.6 Immunohistochemistry/Immunocytochemistry 87
4.7 Flow Cytometry 88
4.8 Functional Blocking and Stimulation Assays 89

Appendix 91
Glossary 93

5
6
 Antibody Theory
Antibody Theory 1
1.1 An Introduction to Antibodies 1.7 Factors Affecting Antigen-Antibody
1.2 Antigens Reactions
1.3 Epitopes 1.8 Generation of Antibodies
1.4 Antibodies 1.9 Antibody Formats
1.5 Antibody-Antigen Interaction 1.10 Biological Effects of Antibodies
1.6 Nature of Antigen-Antibody Bonds

1.1 An Introduction to Antibodies critical tools for most areas of life science research. The
During the first half of the 20th century, a series of basic principle of any immunochemical technique is that
scientific discoveries resolved that antibody-mediated a specific antibody will combine with its specific antigen
immunity is the cornerstone of the specific immune to generate an exclusive antibody-antigen complex. In
response. Since their first use as immunolabeling the following pages we will discuss the nature of this
research tools in the early 1970s, antibody technologies bond, and the use of this robust and specific binding as a
have vastly improved, and antibodies have become molecular tag for research.

1.2 Antigens A variety of molecules such as drugs, simple sugars,

The term antigen is derived from antibody generation, amino acids, small peptides, phospholipids, or
referring to any substance that is capable of eliciting triglycerides may function as haptens. Thus, given
an immune response (e.g., the production of specific enough time, just about any foreign substance will be
antibody molecules). By definition, an antigen (Ag) is identified by the immune system and evoke specific
capable of combining with the specific antibodies formed antibody production. However, this specific immune
by its presence. response is highly variable and depends much in part on
the size, structure, and composition of antigens. Proteins
Generally, antigens are foreign proteins or their or glycoproteins are considered as the most suitable
fragments that enter host body via an infection. antigens due to their ability to generate a strong immune
However, in some cases, the body’s own proteins may response; in other words, they are strongly immunogenic.
act as antigens and induce an autoimmune response.
Bacteria and viruses contain antigens, either on their Antigens are recognized by the host body by two distinct
surface, or inside. These antigens can be isolated and processes (1) by B cells and their surface antibodies
used to develop vaccines. (sIgM) and (2) by the T cell receptor on T cells. Although
both B and T cells respond to the same antigen, they
Antigens are generally of high molecular weight, and respond to different parts of the same molecule.
commonly are proteins or polysaccharides. Polypeptides, Antibodies on the surface of B cells can recognize the
lipids, nucleic acids, and many other materials can also tertiary structure of proteins. On the other hand, T cells
function as antigens. Immune responses may also be require antigens that have been ingested and degraded
generated against smaller substances, called haptens, into recognizable fragments by the antigen-presenting
if these are chemically coupled to a larger carrier cells. Commonly employed antigen-presenting cells are
protein, such as bovine serum albumin, keyhole limpet macrophages and dendritic cells. The immune response
hemocyanin (KLH), or other synthetic matrices. is illustrated in Figure 1. For greater detail on the natural
process of antibody production, a suitable immunology
textbook should be consulted.

7
Antibody Theory

Bacteria Antigen Receptor Characteristics of a Good Antigen

• Areas of structural stability and chemical
B Cell
Processed complexity within the molecule
Antigen Fragment
Macrophage • Significant stretches lacking extensive
repeating units
Antigen Fragment
Receptor Activated • A minimal molecular weight of 8,000 to 10,000
Helper B Cell Daltons, although haptens with molecular weights
T-cell
as low as 200 Da have been used in the presence
of a carrier protein

• The ability to be processed by the immune system

Activated Memory B Cell Antibody-
T-cell Producing • Immunogenic regions that are accessible to the
B Cell
antibody-forming mechanism

• Structural elements that are sufficiently different

Infected Antibody
from those present in the host
Cell
Antigen- • For peptide antigens, regions containing at least
Antibody
Complexes 30% of immunogenic amino acids: K, R, E, D, Q, N
Cytotoxic Memory
T Cell T Cell
• For peptide antigens, significant hydrophilic or
charged residues
Cell-Mediated Immunity Humoral Immunity

Figure 1.
The Immune Response.

1.3 Epitopes
The small site on an antigen to which a complementary
If the target molecule is denatured, e.g., through fixation,
antibody may specifically bind is called an epitope
reduction, pH changes, or during preparation for gel
or antigenic determinant. This is usually one to six
electrophoresis, the epitope may be altered and this
monosaccharides or five to eight amino acid residues on
may affect its ability to interact with an antibody. For
the surface of the antigen. Because antigen molecules
example, some antibodies are ineffective in Western
exist in space, the epitope recognized by an antibody may
blotting (WB) but are suitable for immunohistochemistry
be dependent upon the presence of a specific three-
(IHC) applications, because, in the IHC procedure, a
dimensional antigenic conformation (e.g., a unique site
complex antigenic site might be maintained in the tissue,
formed by the interaction of two native protein loops or
whereas in the WB procedure, the process of sample
subunits). This is known as a conformational epitope. The
preparation alters the protein conformation sufficiently
epitope may also correspond to a simple linear sequence
to destroy the antigenic site, and hence eliminates
of amino acids and such epitopes are known as linear
antibody binding.
epitopes.

The range of possible binding sites on a target molecule

(antigen) is enormous, with each potential binding
Watch Out
site having its own structural properties derived A very important target, αVβ3 integrin will
from covalent bonds, ionic bonds, hydrophilic, and not work in Western blotting experiments
hydrophobic interactions. Indeed, this has important because the epitope is formed by the proximal
ramifications for antibody choice and performance. For association of the αV and β3 subunits to
efficient interaction to occur between the target antigen each other—a conformation destroyed in the
and the antibody, the epitope must be readily available electrophoresis protocol.
for binding.

8
 Antibody Theory
In a denatured protein, only the linear epitope may Knowledge about the target protein, the epitope
be recognized. Hence, in protocols where a denatured recognized by the antibody, sequence conservation, and
protein is used, such as in Western blotting, an antibody the technique principles are valuable in making good
that recognizes a linear epitope is preferred. Sometimes antibody and protocol choices. Actual epitope mapping
an epitope is on the interior of a folded protein. The or sequence data, though useful, are not needed,
epitope is then inaccessible to the antibody in a non- however, to be confident in antibody specificity (see
denaturing protocol, such as immunoprecipitation. A Publishing with Antibodies, section 2.7).
conformational epitope, by definition, is on the outside
R1

of the folded protein. An antibody that recognizes C H

H C
the conformational epitope is suitable for mild, non- N N O
H O C C
denaturing procedures, such as immunoprecipitation or
R2
flow cytometry.

Optimally, an antibody that recognizes a linear epitope

on the surface of a normally folded protein will work well
in both nondenaturing and denaturing protocols.

Thus, the epitope may be present in the antigen’s native, Figure 2.

Amino acids forming a
cellular environment, or it may be exposed only when protein.
denatured. In their natural form, antigens may be
cytoplasmic (soluble), membrane-associated, or secreted.
The number, location and size of the epitopes depend
on how much of the antigen is presented during the
antibody-making process.

1.4 Antibodies number of F(ab) regions on the antibody corresponds

An antibody is defined as “an immunoglobulin capable with its subclass (see below), and determines the valency
of specific combination with the antigen that caused of the antibody (loosely stated, the number of “arms”
its production in a susceptible animal.” Antibodies with which the antibody may bind its antigen).
are produced in response to the invasion of foreign
molecules in the body. An antibody, abbreviated as Ab, Antigen
is commonly referred to as an immunoglobulin or Ig. Binding Site

Human immunoglobulins are a group of structurally and

-
-S

functionally similar glycoproteins (82-96% protein and

-S
-S

-S
-

4-18% carbohydrate) that confer humoral immunity.

-
-S

-S
-S

-
-S

-S
-S
-

-S

-S
-

-S
-S
-
-S

-S-S-
-S

Structure
-S

-
-S

-S
-S

-S-S-
-

Antibodies exist as one or more copies of a Y-shaped

S S
unit, composed of four polypeptide chains. Each Y S S

contains two identical copies of a heavy chain and

Light Chains S S
two identical copies of a light chain, named as such by Heavy Chains S S

their relative molecular weights. This Y-shaped unit is

Variable Region
composed of the two variable, antigen-specific F(ab) F(ab’)2 Fragment
arms, which are critical for actual antigen binding, and F(ab) Fragment
Fc Fragment
the constant Fc “tail” that binds immune cell Fc receptors
and also serves as a useful “handle” for manipulating the Figure 3.
Antibody Structure.
antibody during most immunochemical procedures. The

9
Antibody Theory

These three regions can be cleaved into two F(ab) and The subclasses of antibodies differ in the number of
one Fc fragments by the proteolytic enzyme, papain, disulfide bonds and the length of the hinge region. The
or into just two parts: one F(ab’)2 and one Fc at the most commonly used antibody in immunochemical
hinge region, by pepsin. Fragmenting IgG antibodies procedures is of the IgG class because this is the major
is sometimes useful because F(ab) fragments will not immunoglobulin class released in serum.
precipitate the antigen, and will not be bound by immune
cells in live studies because of the lack of an Fc region. IgA: In the blood IgA are present in low levels in
monomeric form. They are most active at mucosal
surfaces where they are present in dimeric form and

Nice to know provide the primary defense at mucosal surfaces. More

IgA is produced in mucosal linings than all other types
Direct-conjugated antibodies are labeled with of antibody combined. Its major function is to act as
an enzyme or fluorophore in the Fc region. a neutralizing antibody. High levels of IgA are present
The Fc region also anchors the antibody in saliva, tears, and breast milk. In humans two IgA
to the plate in ELISA procedures and is subtypes are known to exist whereas in mice only one
also recognized by secondary antibodies in form is reported. IgA1 may account up to 85% of the
immunoprecipitation, immunoblots, and total IgA in serum. Selective IgA deficiency is one of
immunohistochemistry. the most common immunodeficiency diseases that
increases susceptibility to infections. IgA deficiencies are
Often, because of their smaller size and commonly seen in patients with autoimmune diseases
lack of crosslinking (due to loss of the Fc and allergic disorders. IgA has a half-life of about 5 days.
region), F(ab) fragments are labeled for use
in functional studies. Interestingly, the Fc IgD: It is a monomeric antibody with two epitope
fragments are often used as blocking agents in binding sites and is found on the surface of most B
histochemical staining. lymphocytes. Its precise function is still disputed, but
is suggested to acts as an antigen receptor required
for B cell activation. IgD is also reported to bind to
Subclasses basophils and mast cells and activate them to produce
Antibodies can be divided into five classes: IgG, IgM, IgA, antimicrobial factors. It s also believed to play a role in
IgD, and IgE, based on the number of Y units and the eliminating B-lymphocytes that produce self-reactive
type of heavy chain. Heavy chains of IgG, IgM, IgA, IgD, autoantibodies. IgD is also produced in a secreted form
and IgE, are known as g, µ, a, d, and e, respectively. The that is found in serum in small quantities and contains
light chains of any antibody can be classified as either a two heavy chains of the δ class and two light chains. IgD
kappa (κ) or lambda (λ) type (based on small polypeptide has a half life of about 3 days.
structural differences); however, the heavy chain
determines the subclass of each antibody.

Table 1: Immunoglobulin Subclasses

Class/Subclass Heavy Chain Light Chain MW (kDa) Structure Function
IgA1 a1 l or k 150 to Monomer to tetramer Most produced Ig; protects mucosal surfaces;
IgA2 a2 600 resistant to digestion; secreted in milk
IgD d l or k 150 Monomer Function unclear; works with IgM in B-cell
development; mostly B cell bound
IgE e l or k 190 Monomer Defends against parasites; causes allergic reactions
IgG1 g1 l or k 150 Monomer Major Ig in serum; good opsonizer; moderate
IgG2a g2 complement fixer (IgG3); can cross placenta
IgG2b g2
IgG3 g3
IgG4 g4
IgM μ l or k 900 Pentamer First response antibody; strong complement fixer;
good opsonizer

10
 Antibody Theory
IgE: This group of antibodies is effective at mucosal
surfaces, blood, and tissues. It is present as monomer Nice to know
consisting of two heavy chains (ε chain) and two light
In a beautiful example of convergent evolution,
chains. The ε chain contains 4 Ig-like constant domains.
cartilaginous fishes and camelid mammals, in
In serum, it is present in low concentrations contributing
addition to light and heavy chain antibodies,
to only about 0.002% of total serum antibodies. Most
also have heavy chain-only versions, a smaller
IgE is tightly bound to its receptors on mast cells and
size that could be exploited as a research tool.
basophils via the Fc region. It plays a crucial role in
IgNAR
hypersensitivity reactions and its production is strictly
controlled by cytokines. IgE has a half-life of about
IgNAR

VH

H
V
2 days.
IgNAR

VH

H
V
IgG: This is the most abundant class of antibodies in

CH1
CH1
VH

H
Figure 4.

V
the blood, comprising up to 80% of the total serum
Heavy chain-only

CCHH12
CCHH12
antibodies. It is present in monomeric form. Four
antibodies.
subclasses of IgG have been described depending on

CH 1
C1
H
their abundance (IgG1>IgG2>IgG3>IgG4) and the subclass A heavy-chain shark antibody

CCHH23
CCHH23
(left) and a heavy-chain
produced is dependent on the type of cytokine present. CH2 camelid antibody (middle)
C2
H
in comparison to a common
IgG1 and IgG3 exhibit high affinity for Fc receptors on

CCHH34
CCHH34
antibody (bottom). Heavy
phagocytes, while IgG2 exhibits very low affinity and IgG4 chains are shown in a darker
CH3
C3

shade, light chains in a

H

has moderate affinity for Fc receptors IgGs are capable lighter shade.
CCHH45
CCHH45
of exiting the circulatory system and enter tissues. IgG1,
CH4
C4
H

IgG3, and IgG4 can cross placental barrier to provide

CH5
CH5

protection for newborns. IgGs are efficient at activating

CH5
C5

the complement system, and are very effective for

H

opsonization using Fc receptors on phagocytes. Through

hcIgG
its Fc region IgG can also bind to natural killer cells and
participate in antibody-dependent cytotoxicity. IgG has
hcIgG
VH

H
V

a half-life ranging from 7 to 23 days, depending on its

subclass. hcIgG
VH

H
V
VH

H
CH2
C2
V

IgM: This class of immunoglobulin is first to be

H

produced in response to infection and is found

CCHH23
CC 23

either on membranes of B cells or as a 5-subunit

HH
CH2
C2

macromolecule secreted by plasma cells. It is also the

H

CH 3
C3

first immunoglobulin class to be synthesized by the

H

neonates. The surface IgM differs from the secreted form

CH3
C3
H

in its Fc region. Surface IgM binds directly as an integral

membrane protein and not to the IgM Fc receptor.
Secreted IgM is a pentameric molecule where multiple IgG
VH

H
V
V
I

immunoglobulins are covalently linked with disulfide

VI

IgG
1
CH

H
C
1

bonds. This structure provides multiple binding sites.

VH

H
CI

C
VI

Each monomer consists of two light chains (either κ IgG

V
I
VI

1
VH

H
CH

H
C

or λ) and two heavy chains. Because of its pentameric

1
CH2
V CH2
I
VI

CI

C
I
1
CH

nature IgM is particularly suited for activating

H
C
1
CI

C
I

complement and causing agglutination. IgM has a

CCHH23
CCHH23

half-life of about 5 days.

CH2
CH2

CH3
CH3
CH3
CH3

11
Antibody Theory

Antigen
1.5 Antibody-Antigen Interaction Immunochemical techniques capitalize upon the
Now that you know what an antigen and antibody extreme specificity, at the molecular level, of each
Antigen
are, let us consider the interaction between them. The immunoglobulin for its antigen, even in the presence of
Binding Site
strength of interaction between antibody and antigen high levels of contaminating molecules. The multivalency
at single antigenic sites can be described by the affinity of most antigens and antibodies enables them to
of the antibody for the antigen. Within each antigenic interact to form a precipitate. Examples of experimental
Antibody
site, the variable region of the antibody “arm” interacts applications that use antibodies are Western blot,
through weak noncovalent forces with antigen at immunohistochemistry and immunocytochemistry,
numerous sites. The greater the interaction, the stronger enzyme-linked immunosorbent assay (ELISA),
the affinity. Avidity is perhaps a more informative immunoprecipitation, and flow cytometry. Each is
measure of the overall stability or strength of the discussed in more detail in later sections of this reference
antibody-antigen complex. It is controlled by three major guide.
factors: antibody epitope affinity, the valence of both the
antigen and antibody, and the structural arrangement Antibody-Antigen Interaction Kinetics
of the interacting parts. Ultimately these factors define The specific association of antigens and antibodies is
the specificity of the antibody, that is, the likelihood that dependent on hydrogen bonds, hydrophobic interactions,
the particular antibody is binding to a precise antigen electrostatic forces, and Van der Waals forces. These
epitope. are of a weak, noncovalent nature, yet some of the
associations between antigen and antibody can be quite
Cross-reactivity refers to an antibody or population of strong. Like antibodies, antigens can be multivalent,
antibodies binding to epitopes on other antigens. This either through multiple copies of the same epitope,
can be caused either by low avidity or specificity of the or through the presence of multiple epitopes that are
antibody or by multiple distinct antigens having identical recognized by multiple antibodies. Interactions involving
or very similar epitopes. Cross-reactivity is sometimes multivalency can produce more stabilized complexes;
desirable when one wants general binding to a related however, multivalency can also result in steric difficulties,
group of antigens or when attempting cross-species thus reducing the possibility for binding. All antigen-
labeling when the antigen epitope sequence is not highly antibody binding is reversible and follows the basic
conserved during evolution. Cross-reactivity can result in thermodynamic principles of any reversible bimolecular
over- or under-estimation of the antigen concentration interaction:
and is problematic in immunoassays.

[Ab-Ag]
KA =
[Ab][Ag]
Nice to know
Researchers working with Drosophila, Xenopus,
zebrafish, and other non-mammalian model
where KA is the affinity constant, [Ab-Ag] is the molar
organisms are often faced with antibodies
concentration of the antibody-antigen complex, and [Ab]
validated only in mammals. Choosing
and [Ag] are the molar concentrations of unoccupied
polyclonal antibodies made against whole
binding sites on the antibody (Ab) or antigen (Ag),
fusion proteins, or larger and conserved
respectively.
immunogen sequences, provides the best
chances for interspecies cross-reactivity.

12
 Antibody Theory
The time taken to reach equilibrium is dependent on the be made by equilibrium dialysis. Repeated equilibrium
rate of diffusion and the affinity of the antibody for dialyses with a constant antibody concentration, but
the antigen and can vary widely. The affinity constant varying ligand concentration are used to generate
for antibody-antigen binding can span a wide range, Scatchard plots, which give information about affinity
extending from below 10 /mol to above 10 /mol.
5 12
valence and possible cross-reactivity.
Affinity constants can be affected by temperature, pH,
and solvent. Affinity constants can be determined for When designing experimental procedures, it is important
monoclonal antibodies, but not for polyclonal antibodies, to differentiate between monoclonal and polyclonal
as multiple bond formations take place between antibodies, as these differences are the foundation of
polyclonal antibodies and their antigens. Quantitative both advantages and limitations of their use.
measurements of antibody affinity for antigen can

1.6 Nature of Antigen- • The specific binding between the antigenic determinant
on the cell (known as epitope) and the antigen-
Antibody Bonds
combining site (paratope) on the antibody involves very
The combining site of an antibody is located in the F(ab)
small portions of the molecules, usually comprising only
portion of the antibody molecule and is assembled from
a few amino acids.
the hypervariable regions of the heavy and light chains.
• These sites are critical in antigen-antibody reactions as
The binding between this site and the antigen takes place
specific binding has to overcome repulsion between the
with the following characteristics and processes:
two molecules.
• The bonds that hold the antigen to the combining site
• When the epitope comes in contact with paratope
of any antibody are noncovalent, and, hence, they are
they are first attracted to each other by ionic and
reversible in nature.
hydrophobic forces.
• These bonds may be hydrogen bonds, electrostatic
• These forces help them overcome their hydration
bonds, or Van der Waals forces.
energies and allow for the expulsion of water molecules
• Usually there are multiple bond formations observed,
as epitope and paratope approach each other.
ensuring relatively tight binding between antibody and
• This attraction becomes even stronger when Van der
antigen.
Waals forces are employed later on to bring epitope and
paratope even closer.

1.7 Factors Affecting Antigen- antibody reaction is strongly inhibited. At pH 5.0 or 9.5,
the equilibrium constant is 100-fold lower than at pH
Antibody Reactions
6.5 - 7.0. Under extreme pH conditions, antibodies may
The antigen-antibody reaction can be influenced by
undergo conformational changes that can destroy the
several factors. Some of the more common factors are:
complementarity with the antigen.

Temperature
Ionic strength
The optimum temperature for antigen-antibody reaction
Effect of ionic strength on antigen-antibody reaction
will depend on the chemical nature of the epitope,
is particularly important in blood group serology. Here
paratope, and the type of bonds involved in their
the reaction is significantly influenced by sodium and
interaction. For example, hydrogen bond formation tends
chloride ions. For example, in normal saline solution,
to be exothermic. These bonds are more stable at lower
Na+ and Cl− cluster around the complex and partially
temperature and may be more important when dealing
neutralize charges, potentially interfering with antibody
with carbohydrate antigens.
binding to antigen. This could be problematic when
low-affinity antibodies are used. It is well known
pH
that, when exposed to very low ionic strengths,
The effect of pH on the equilibrium constant of the
γ-globulins aggregate and form reversible complexes
antigen-antibody complex lies in the pH range of 6.5
with lipoproteins of red blood cells, leading to their
and 8.4. Below pH 6.5 and above pH 8.4, the antigen-
sedimentation.
13
Antibody Theory

Nice to know
Prozone effect is a phenomenon when in an antibody-antigen laboratory testing false negative or false low
results occur from the excess of antigen or antibody in a sample due to the inability of the analyte to bind
to receptor sites.

In a typical immunoassay, antigens and antibodies bind to create a conjugate that can be detected and
measured. However, when prozone effect occurs, excess antigens or antibodies can bind all of the receptor
sites, leaving no molecules available to form conjugates. Hence, antibody-antigen conjugates cannot be
detected and a false negative result is produced, which can go undetected. In a clinical setting, this could
lead to misdiagnosis. If an error in results is suspected, one should always dilute the sample and retest.

1.8 Generation of Antibodies mixtures of antibodies react with multiple epitopes on

the surface of the antigen, they will be more tolerant
Polyclonal and Monoclonal Antibodies
of minor changes in the antigen, e.g., polymorphism,
Antibodies are normally produced by B cells, which
heterogeneity of glycosylation, or slight denaturation,
are part of the immune system, in response to the
than will monoclonal (homogenous) antibodies.
introduction of foreign substances, such as infectious
agents, into the animal’s body. The antibodies bind to the
Depending upon the antigen that is used to create the
antigens that cause their generation and flag them for
antibody, one may use polyclonal antibodies to identify
destruction, thus helping to fight infection. This inherent
proteins of high homology to the immunogen protein or
ability of the animal’s body can be leveraged to generate
to screen for the target protein in tissue samples from
antibodies that bind to specific molecules. Target-specific
species other than that of the immunogen. Along the
antibodies can be used to isolate and identify molecules
same lines, it is especially important when working with
of interest. Antibodies have become one of the most
polyclonal antibodies to learn as much as possible about
important tools in life science research, allowing the
the immunogen that has been used for production of
detection, quantitation, and determination of changes
the polyclonal antibody and the potential for undesired
in proteins and other molecules with respect to time and
cross-reactivity within the sample being analyzed.
other perturbations.
Peptide immunogens are often used to generate
polyclonal antibodies that target unique epitopes,
Many of the antibodies used in immunochemical
especially for protein families of high homology.
techniques are raised by repeated immunization of a
suitable animal, e.g., rabbit, goat, donkey, or sheep, with
an appropriate antigen. Serum is harvested at the peak
Large antigen immunogen
of antibody production. Specific IgG concentrations of yields multiple epitope
polyclonal antibodies
approximately 1 to 10 mg/mL serum can be obtained by
this method. Weakly antigenic molecules may require
the addition of an adjuvant, which allows for the slow
release of the antigen, making it more readily trapped by
macrophages. Smaller molecules, such as drugs, must be Small peptide immunogen
yields fewer, restricted epitope
coupled to more antigenic structures (i.e. carrier proteins) polyclonal antibodies
Each B cell
to stimulate an immune response. only produces
antibodies to
one epitope Isolate and fuse B cell to
hybridoma line & screen
One characteristic of large antigen molecules is that they
induce the activation of many antibody-producing B cell Monoclonal antibody is
clones in the immunized animal. This polyclonal mixture restricted to only one epitope

of resulting antibodies may then recognize a variety of

epitopes on the antigen, which can be a useful feature in
Figure 5.
some experimental procedures. Because these polyclonal Production of monoclonal vs. polyclonal antibodies.
14
 Antibody Theory
A homogeneous population of antibodies (i.e. diagnostic applications because monoclonal antibodies
monoclonal antibodies) can be raised by fusion of B react with one epitope on the antigen. However, they are
lymphocytes with immortal cell cultures to produce more vulnerable to the loss of epitope through chemical
hybridomas. Hybridomas will produce many copies of the treatment of the antigen than are polyclonal antibodies.
exact same antibody. This impressive phenomenon has This can be offset by pooling two or more monoclonal
been instrumental in the development of antibodies for antibodies to the same antigen.

Some Useful Properties of Some Useful Properties of

Polyclonal Antibodies Monoclonal Antibodies
• Polyclonal antibodies often recognize multiple • Because of their specificity, monoclonal antibodies
epitopes, making them more tolerant of small are excellent as the primary antibody in an assay,
changes in the nature of the antigen. Polyclonal or for detecting antigens in tissue, and will often
antibodies are often the preferred choice for result in significantly less background signal than
detection of denatured proteins. polyclonal antibodies.

• Polyclonal antibodies may be generated in a variety • When compared to that of polyclonal antibodies,
of species, including rabbit, goat, sheep, donkey, homogeneity of monoclonal antibodies is very high.
chicken, and others, giving the users many options
• If experimental conditions are kept constant,
in experimental design.
results from monoclonal antibodies will be highly
• Polyclonal antibodies are sometimes used when the reproducible between experiments.
nature of the antigen in an untested species is not
• Specificity of monoclonal antibodies makes them
known.
extremely efficient for binding of antigen within a
• Polyclonal antibodies target multiple epitopes and mixture of related molecules, such as in the case of
so they generally provide more robust detection. affinity purification.

Table 2: Advantages and Disadvantages of Polyclonal and Monoclonal Antibodies

Advantages Disadvantages
Relatively easy to generate and more cost-effective. Animal death can terminate the source of antibody.
Multiple epitopes on the same protein can generate many antibodies. Different bleeds may give different results.
Hence, they provide more robust signals.
Polyclonal Antibodies

Polyclonal antibodies can generate better signals with proteins expressed in Immunization of a new animal with the same antigen may lead to different
low levels. epitopes and different clones may be generated.
They are compatible with a broader range of applications. Shared epitopes on different proteins can lead to labeling of proteins other
than the antigen protein.
Polyclonal antibodies provide more flexibility in antigen recognition. For Greater batch-to-batch variability is possible.
example, they may bind the antigen in spite of polymorphism, heterogeneity
of glycosylation etc. Hence, they can identify proteins of high homology or
from different species.
Better suited for the detection of denatured proteins. May produce nonspecific antibodies that can add to background signal.
Different clones of antibodies can be generated to different epitopes on a Production of monoclonal antibodies is more labor-intensive. More work is
single antigen. required, especially in the cloning and selection process.
Monoclonal Antibodies

Hybridoma cells can serve as an infinite source of the same antibody. They may be limited in their applications.
The high specificity of monoclonal antibodies minimizes background and A vast majority of monoclonal antibodies are produced in mice because of a
eliminates cross-reactivity. robust myeloma cell line.
Their homogeneity is very high and they provide consistent, reproducible High specificity of monoclonal antibodies limits their use in multiple
results. species.
They bind only to one antigen in a mixture of related proteins. Monoclonal antibodies are more susceptible to the loss of epitope through
chemical treatment of the antigen.
Batch–to-batch variability is very minimal.

15
Antibody Theory

Clone Numbers • Polyclonal antibodies contain multiple clones of

Each clone number represents a specific cell line that antibodies produced to different epitopes on the
was used to produce the antibody. Since antibodies are antigen. For example, if there are four epitopes on the
produced by more than one host, each cloned cell line antigen then four different clones of antibodies will
receives a unique clone number. Each hybridoma cell be produced.
clone produces only one single pure antibody type. • Different antibody clones may have different
properties and may even be of different isotypes.
• An animal injected with an antigen will generate They may also work in different applications. Hence,
multiple antibodies to many epitopes. Since it is best to select an antibody clone that will work
antibodies are produced by B cells, a single clone of B optimally in your choice of application.
cells can produce antibodies to only a single epitope. • It is important to recognize that a clone number is
• Monoclonal antibodies are derived from a single clone not synonymous with the lot number, which often
of cells and can be generated in larger quantities. indicates the date of manufacture.

1.9 Antibody Formats Antigen affinity purification takes advantage of the affinity

As the name implies, the antibody format refers to the of the specific immunoglobulin fraction for the immunizing

presentation or purification state of the antibody. Various antigen against which it was generated. This method may

formats are described below: be used to remove unwanted antibodies from a preparation.
The preparation of antibodies is passed through a column

Polyclonal antibodies are often available in relatively matrix containing antigens against which the unwanted

unpurified formats, and are referred to as “antiserum” or antibodies are directed. The unwanted antibodies remain

simply as “serum”. Antiserum refers to the blood from bound to the column, and the effluent contains the desired,

an immunized host from which the clotting proteins and affinity-purified antibodies. Alternatively, a column matrix

RBCs have been removed. The antiserum, as its name coupled to the desired antigen can be used. In this case,

suggests, still possesses antibodies/immunoglobulins of antibody directed against the coupled antigen remains

all classes as well as other serum proteins. In addition to bound to the column and may be then eluted using a

antibodies that recognize the target antigen, the antiserum solution that disrupts antigen-antibody binding. Unlike

also contains antibodies to various other antigens that protein A/G purification, antigen affinity purification

can sometimes react nonspecifically in immunological results in the elimination of the bulk of the nonspecific

assays. For this reason, raw antiserum is often subjected immunoglobulin fraction, while enriching the fraction of

to purification steps, to eliminate serum proteins and to immunoglobulin that specifically reacts with the target

enrich the fraction of immunoglobulin that specifically antigen. The resulting affinity purified immunoglobulin will

reacts with the target antigen. contain primarily the immunoglobulin of desired specificity.

Antiserum is commonly purified by one of two Typically, affinity purified antibodies exhibit lower

methods: Protein A/G purification or antigen affinity backgrounds than unabsorbed antibodies and this

chromatography. purification process is particularly important for difficult,

or state-dependent epitopes. When developing polyclonal

Protein A/G purification takes advantage of the antibodies that recognize targets with post-translational

high affinity of Staphylococcus aureus protein A or modifications, the use of modification specific antigen

Streptococcus protein G for the immunoglobulin Fc affinity columns during the purification process can

domain. While protein A/G purification eliminates the bulk significantly improve the specificity of the antibody for

of the serum proteins from the raw antiserum, it does not state-dependent target. Depleting unmodified target

eliminate the nonspecific immunoglobulin fraction. As a protein from the serum before affinity purification

result, the protein A/G purified antiserum may still possess (using immobilized, modified target protein) increases

undesirable cross reactivity. See Protein A/G Binding the specificity for the modified target. Specificity testing

Affinities in Appendix. can then be performed to confirm that the antibody only
recognizes the post-translationally modified form of the
protein.

16
 Antibody Theory
Monoclonal antibodies may be grown in cell cultures is unknown, one may refer to the following “typical
and collected as hybridoma supernatants, or grown in ranges” as a guideline for estimation: Tech Tip
mice or rats and collected as relatively unpurified ascites
Antibody
fluid. These can be purified through the use of protein Polyclonal Antiserum: Specific antibody concentrations
concentrations
A/G or specific antigen affinity chromatography as with will typically range from 1–3 mg/mL.
of purified
polyclonal antibodies. Hybridoma Supernatant: Specific antibody
preparations should
concentrations will typically range from 0.1–10.0 mg/mL.
be determined prior
Unpurified antibody preparations vary significantly in Ascites Fluid (unpurified): Specific antibody
to the addition of
specific antibody concentration. If the specific antibody concentrations will typically range from 2–10 mg/mL.
stabilizing protein
concentration of a given unpurified antibody preparation
such as BSA.

1.10 Biological Effects of Antibodies Cytolysis

Antibodies are widely used for protection from infectious Certain antibodies can cause disruption of the microbial
agents. Most vaccines (microbial antigens) induce the membrane that result in death of bacterial cells. This
production of antibodies that block infection or interfere requires the participation of the complement system.
with microbial invasion of the bloodstream. To achieve this,
antibodies must be functional in the sense that they are Opsonization
capable of neutralization or opsonophagocytosis. In this process, the pathogenic organism is targeted for
digestion by phagocytes. The antibody binds to a receptor
The membrane attack complex on the cell membrane of the bacterium, attracting
(MAC) cytolysis phagocytes to the site. The F(ab) portion of the antibody
MAC is formed on the surface of pathogenic bacterial binds to the antigen, while the Fc portion of the antibody
cell as a result of the activation of the complement binds to an Fc receptor on the phagocyte, facilitating
system (both alternative and the classical pathways). The phagocytosis. This process is further enhanced by the
MAC forms transmembrane channels in bacterial walls, complement system.
disrupting their phospholipid bilayer and leading to cell
lysis and death. Neutralization of exotoxins
Antitoxin antibodies can be generated against microbial
Neutralization of viruses toxins. The F(ab) region of the antibody made against
Antibodies can interfere with virion binding to receptors epitope of the binding site of an exotoxin can block the
and block their uptake into cells. Many enveloped viruses exotoxin from binding to the exotoxin receptor on the host
are lysed when antiviral antibodies and the complement cell membrane. This blocks the entry of the toxin into the
system disrupt membranes. Certain antibodies can also cell.
aggregate virus particles. Non-neutralizing antibodies
are also produced following any viral infection. Although Preventing bacterial adhesion to host cells
these antibodies bind specifically to virus particles, they do The body’s innate defenses can physically remove bacteria
not neutralize them. On the contrary, they may enhance by constant shedding of surface epithelial cells from the
infectivity because the virus-antibody complex enters the skin and mucous membranes. However, bacteria may
cell by endocytosis. This can lead to viral replication. resist this by producing pili, cell wall adhesin proteins,
and biofilm-producing capsules. The F(ab) region of the
The type of antibody produced can influence the outcome antibody can bind to the adhesive tip of the pili, the cell
of viral infection. For example, poliovirus can elicit IgM wall adhesins, or the capsular molecules, and blocks
and IgG responses in the blood, but mucosal IgA is vital bacterial adhesion to host cells.
for blocking infection. The IgA neutralizes poliovirus in
the intestine, the site of primary infection. Hence, the Agglutination of microorganisms
live attenuated Sabin poliovirus vaccine is more effective The F(ab) sites of IgM and IgA antibodies can link
because it elicits a strong mucosal IgA response. microorganisms together and cause them to agglutinate.
The agglutinated microorganisms can be phagocytosed
Immobilization more effectively.
An antibody can be directed against cilia or flagella of motile
bacteria or protozoa that results in cessation of their motility
and blocks their ability to move around and spread infection.

17
Antibody Theory

Technology Highlight

Dot blot arrays for testing antibody specificity

Antibody specificity is critical when performing chromatin immunoprecipitation or other sensitive antibody-dependent analyses. The
diversity of post-translational modifications to histone protein targets makes unreliable antibody specificity and precision major sources
of variation and error in data interpretation. Most antibodies are not tested to determine cross-reactivity among various modifications;
however, even small changes in protein state, like dimethyl to trimethyl labeling, have important biological implications. Developing dot
blot arrays is an effective way to screen antibodies for specificity.

AbSurance™ Histone Antibody Specificity Arrays employ the same technology used to screen EMD Millipore’s highly characterized
and extensively published histone antibodies. Built on easy-to-use Immobilon®-FL PVDF membranes, these arrays permit a detailed
characterization of antibodies against key histone modification sites. The AbSurance™ screening process is performed using a simple,
Western-blot–like procedure, followed by detection using either X-ray film or a CCD imager.

A. Acetyl Histone H4 (Lys12)

1 2 3 4 5 6 7 8 9 10 11 12

A AbSurance™ Benefits
B • High quality purified peptides
(>95% purity)
C
• 89 peptides representing all key
D histone modification sites (acetyl,
phospho, and mono-, di-, and
E trimethyl PTMs)

F • Consistent and uniform spotting

of peptides using a proprietary
G process

H • Sensitive chemiluminescent
detection using either film or CCD
imagers
B. Location of reactivity with the H4 antibody and Control IgG Tested
• Easy data analysis—no additional
software required 1 2 3 4 5 6 7 8 9 10 11 12
H2A H2A H2A H2A H2A H2A H2A H2A.X H2A.X H2A.X H2B H2B
• Built-in positive control primary A 1-19 1-19 1-19 1-19 1-19 110-129 110-129 124-142 124-142 124-142 1-19 1-19 100 ng
unmod SIP K5ac K9ac K13ac unmod T120P unmod S139P Y142P unmod K5ac
antibodies from rat, mouse, sheep, H2A H2A H2A H2A H2A H2A H2A H2A.X H2A.X H2A.X H2B H2B
B 1-19 1-19 1-19 1-19 1-19 110-129 110-129 124-142 124-142 124-142 1-19 1-19 10 ng
and rabbit unmod SIP K5ac K9ac K13ac unmod T120P unmod S139P Y142P unmod K5ac
H2B H2B H2B H2B H2B H2B H4 H4 H4 H4 H4 H4
C 1-19 1-19 1-19 1-19 107-125 107-125 7-26 7-26 7-26 7-26 7-26 7-26 100 ng
K5me1 K12ac S14P K15ac unmod K120ac unmod SIP R3me1 R3me2a R3me2s K5ac
H2B H2B H2B H2B H2B H2B H4 H4 H4 H4 H4 H4
D 1-19 1-19 1-19 1-19 107-125 107-125 7-26 7-26 7-26 7-26 7-26 7-26 10 ng
K5me1 K12ac S14P K15ac unmod K120ac unmod SIP R3me1 R3me2a R3me2s K5ac
H4 H4 H4 H4 H4 H4 H4 H4 H4 H4 H4 H4
E 1-19 1-19 11-30 11-30 11-30 11-30 11-30 11-30 11-30 11-30 11-30 11-30 100 ng
K8ac K12ac unmod K16ac R17me1 R17me2a R17me2s R19me1 R19me2a R19me2s K20ac K20me1
H4 H4 H4 H4 H4 H4 H4 H4 H4 H4 H4 H4
F 1-19 1-19 11-30 11-30 11-30 11-30 11-30 11-30 11-30 11-30 11-30 11-30 10 ng
K8ac K12ac unmod K16ac R17me1 R17me2a R17me2s R19me1 R19me2a R19me2s K20ac K20me1
H4 H4 H4 H4 H4 H4 H4
Rat Sheep
G 11-30 11-30 11-30 11-30 11-30 82-100 82-100 100 ng
IgG IgG 100 ng
K20me2 K20me3 R23me1 R23me2a R23me2s unmod K19ac
H4 H4 H4 H4 H4 H4 H4
Mouse Rabbit
H 11-30 11-30 11-30 11-30 11-30 82-100 82-100 10 ng
IgG IgG 10 ng
K20me2 K20me3 R23me1 R23me2a R23me2s unmod K19ac

Specificity screening of histone H4 antibody.

A. The Histone H2A, H2B, H4 Array was probed with anti-acetyl histone H4 (Lys12) antibody
(1:2000 dilution, Cat. #04-119). Peptides were visualized using a donkey anti-rabbit IgG,
peroxidase conjugated, H+L (Cat. #AP182P) secondary antibody and a chemiluminescence de-
tection system. B. Peptide map showing location of reactive peptide spots (Dark Blue). Control
rabbit IgG is shown in lighter shade of blue.

18
Notes

19
20
 ANTIBODY PRACTICE
Antibody Practice 2
2.1 Selection and Use 2.5 Use of Secondary Antibodies
2.2 Antibody Titer and Concentration 2.6 Proper Controls
2.3 Storage and Handling of Antibodies 2.7 Publishing with Antibodies
2.4 Conjugated Antibodies 2.8 Validating Antibodies

2.1 Selection and Use Species from which the protein is to be detected:
Considerations when selecting an antibody • Select an antibody that is raised against the
for use in an experiment immunogen sequence derived from species of your
Once you have identified your target antigen and have interest.
chosen your detection method, you must then choose • If the sequence is not derived from your species of
one or more primary antibodies to detect your target. interest, check to see if it will react with your sample.
If more than one potential antibody is available for You may quickly check the sequence for specific
your target, it may be recommended to carry out key proteins in the protein data bank: http://www.ncbi.
experiments using multiple antibodies (see section 2.7 nlm.nih.gov/protein.
on Publishing with Antibodies). Choose your antibodies
based on the following considerations: Species in which the antibody is raised:
• This information will be of great advantage when
Determine the best application for your selecting a secondary antibody. The secondary
research need: antibody should be phylogenetically as far apart as
• Not all antibodies will work with every application. possible from a species from which your sample is
• Determine if you are performing a qualitative or derived.
quantitative assay
• Check vendor’s data sheet or website to see if the Check for validation data available on data sheet or
antibody is suitable for the specific application, such vendor website:
as immunoblotting, ELISA etc. • Look at the validation data on data sheet or on
vendor’s website and examine the quality of data.
Type of sample being tested: • Check to see if only a verification of the presence
• Does your tissue or cell express the particular protein? of antigen is provided (ELISA, Western blotting) or
• Are you trying to detect a latent or activated protein? whether there are other in-depth data.
For example, phospho-specific antibodies may react • Check to see what type of sample was tested (cell
only with activated phosphorylated proteins. lysate, tissue homogenate etc). Just using purified
• If your protein has an intracellular location it will be recombinant protein may not give best results with
necessary to perform a cell lysis. real cell or tissue samples.
• In flow cytometric analysis it may be necessary to use
an antibody that recognizes cell surface molecules. Guarantee and support:
• If your protein has a tertiary structure and the epitope • Is there an offer of guarantee from the vendor? It may
is obscured then sample has to be denatured because be money-back or credit.
antibody will not recognize the native state. • What type of technical support is available? It is best
• Some antibodies will work best only in frozen or on to have access to live technical support as opposed to
unfixed tissue and others will work in paraffin sections frequently asked questions on the website.
only after an antigen retrieval process.

21
ANTIBODY PRACTICE

2.2 Antibody Titer and tests with minimum background reaction (e.g., for
negative controls). The optimal antibody concentration
Concentration
must be determined experimentally for each assay, and is
The binding of antibody and antigen is dependent on
typically determined by using a dilution series.
the affinity constant, which, in turn, can be affected
by temperature, pH, solvent composition, etc. Varying
The optimal antibody concentration is best determined
the relative concentrations of antibody and antigen in
by first selecting a fixed incubation time and preparing a
solution can also control the extent of antibody-antigen
series of dilutions to test. Dilutions are usually expressed
complex formation.
as the ratio of the more concentrated stock solution to
the total volume of the desired solution. For example, a
Concentration and titer are not equivalent. Concentration
1:10 dilution of antibody is created by mixing one part of
is the total amount of antibody contained in the solution.
antibody stock solution with nine parts of diluent, giving
Usually, only a percentage of it represents the intact,
a total of ten parts.
active, and functional antibody with regard to its ability
to bind the antigen, and determines its effectiveness. The
Datasheets and protocols may suggest approximate
titer is the highest dilution of the antibody that yields a
dilutions for antibody use. When using an antibody
response in the immunoassay. It is the degree to which
for the first time, or when working with a new batch
the antibody-serum solution can be diluted and still
of antibody, it is advisable to try a dilution series to
contain detectable amounts of antibody.
determine the optimal antibody dilution to use. For
example, if a product data sheet suggests using a 1:500
In most cases, the concentration of antigen in a
dilution, making dilutions of 1:50, 1:100, 1:500, 1:1,000
sample cannot be adjusted. Hence, the optimal working
and 1:10,000 can help determine the optimal dilution for
concentration (dilution) of the antibody must be
a set of unique assay conditions. Especially in the case
determined empirically for a given set of experimental
of polyclonal antisera, antibody concentrations may be
conditions.
significantly different from animal to animal or from one
serum bleed to the next, and this kind of initial titration
For any assay, the optimum titer is that concentration
is essential in reducing inter-assay variations.
(dilution) which gives the strongest reaction for positive

2.3 Storage and Handling of • Antibodies are relatively stable proteins and are
resistant to a broad range of mild denaturing
Antibodies
conditions. Most antibodies are stable for years
The proper storage and handling of antibodies is
when stored properly as per manufacturer’s
critical to their function and longevity. Properly stored
recommendations.
antibodies show little degradation over long periods of
• In most cases antibodies can be stored at -20°C
time, extending their usefulness to several months or
without any loss in their binding capacity.
even years. Improperly stored antibodies, on the other
• It is best to avoid storing antibodies in a frost-free
hand, can denature in a matter of hours. Consider the
freezer. This is to avoid or minimize freeze-thaw cycles.
following points when storing and handling antibodies
Antibody solutions should not be frozen and thawed
and other biological reagents:
repeatedly, as this can lead to aggregation, causing
a loss of activity. Hence, stock solutions should be
• In order to preserve maximum reactivity, reagents
aliquoted prior to storage.
should be stored according to the manufacturer’s
• Undiluted antibodies should always be aliquoted prior
instructions (e.g., avoid holding antibodies at room
to storage at -20°C to minimize repeated freeze/thaw
temperature when storage at 2–8°C is indicated).
cycles that can denature antibody. Storing antibody
• It is a good rule of thumb to store antibodies in tightly
in concentrated form will either prevent or minimize
sealed containers in a non-frost-free refrigerator/
degradation. A cryoprotectant, such as glycerol, to
freezer, away from tissue fixatives and crosslinking
a final concentration of 50%, can be added to the
reagents.
antibody solution to prevent freeze/thaw damage. Do
not store glycerol-containing antibodies at -80°C.
22
 ANTIBODY PRACTICE
• Unless a stabilizing protein, such as BSA (1% w/v), azide is toxic. As with all laboratory reagents, consult
has been added, antibodies should not be stored for a Material Safety Data Sheet (MSDS) for handling
extended periods at their working dilutions. Avoid precautions.
storing diluted antibodies for extended periods. • Generally, enzyme-conjugated antibodies are not
• The major problem encountered during storage is frozen to prevent loss of enzyme activity and their
contamination with bacteria or fungi. If antibodies binding capacity. It is best to store them at +4°C.
are stored at 2-8°C for more than two to three days, • Fluorescent conjugates are susceptible to
it is advisable to filter-sterilization and/or add a photobleaching. Hence, fluorochrome-conjugated
bacteriostat/preservative, such as 0.05% sodium azide antibodies should be stored protected from light in a
or 0.1% thimerosal. darker colored vial.
• Sodium azide can interfere with various biological • When stored for a long period of time, some
assays and with some coupling methods. Hence, in antibody solutions may produce an insoluble lipid
these applications it is best to either remove sodium component. The precipitate can be removed by a quick
azide by centrifugal diafiltration, dialysis, or gel centrifugation at 10,000 g.
filtration, or use azide-free antibodies. Note: Sodium

2.4 Conjugated Antibodies different buffers and storage conditions to retain their

A note on concentrations, storage buffers, maximal activity over time. The following table lists
and storage temperatures the standard antibody buffers and storage conditions
for purified EMD Millipore antibodies and antibody
Often for signal amplification and detection purposes,
conjugates. Note that these are general guidelines
purified antibodies are conjugated to enzymes,
and that one should always consult the datasheet
fluorophores, or haptens, such as horseradish peroxidase
accompanying the antibody for specific storage
(HRP), alkaline phosphatase (AP), rhodamine, fluorescein
conditions for that antibody.
isothiocyanate (FITC), or biotin. The various antibody
conjugates have differential stabilities and require

Antibody Buffers Standard Antibody Concentrations

Purified and Monoclonal Conjugates 1 mg/mL
1. Affinity-purified Monoclonal and Polyclonal
Polyclonal Affinity-purified Antibodies 2 mg/mL
Antibodies
Polyclonal FITC Conjugates 2 mg/mL
0.02 M phosphate buffer, 0.25 M NaCl,
Polyclonal HRP/Alk Phos Conjugates 1 mg/mL
0.1% NaN3, pH 7.6
Same buffer without NaN3 may be used as required.

2. FITC Conjugates
0.02 M phosphate buffer, 0.25M NaCl, 15 mg/mL BSA, Antibody Storage Conditions
0.1% NaN3, pH 7.6 Polyclonal Affinity-purified Antibodies 4°C to 8°C
Fluorescent Conjugates (Store in dark) 4°C to 8°C
3. HRP Conjugates Enzyme Conjugates (Do not freeze) 4°C to 8°C
0.01 M PBS, 15 mg/mL BSA, 0.01% Thimerosal, pH 7.1 Hapten Conjugates (Do not freeze) 4°C to 8°C

4. Alkaline Phosphatase Conjugates

0.05 M Tris, 0.1 M NaCl, 0.001 M MgCl2,
15 mg/mL BSA, 0.1% NaN3, pH 8.0

5. Biotinylated Conjugates
0.01 M PBS, 15 mg/mL BSA, 0.1% NaN3, pH 7.1

23
ANTIBODY PRACTICE

2.5 Use of Secondary Antibodies Selecting an appropriate secondary antibody:

Secondary antibodies are often used to indirectly detect • Secondary antibody should be against the same
an antigen to which a primary antibody is first bound. species in which the primary antibody is raised. For
Hence, it is important to select a secondary antibody that example, if the primary antibody is raised in goat then
has specificity for the antibody species and isotype of the secondary antibody should be anti-goat.
primary antibody and is conjugated to a detectable tag or • Select an antibody labeled with a fluorochome or
label for detection. Consider the following points: enzyme of your choice, and your expertise and
the instruments available in your laboratory. More
• The detectable tag could be an enzyme or a commonly used fluorochrome labels are fluorescein,
fluorochrome. Most commonly used tags are rhodamine, Texas Red, phycoerythrin, etc, and enzyme
horseradish peroxidase, alkaline phosphatase, conjugates could be horseradish peroxidase, alkaline
fluorescein isothiocyanate (FITC), rhodamine, Texas phosphatase, etc.
Red, phycoerythrin, and biotin. • Biotin-conjugated antibodies provide greater
• A proper selection of secondary antibody can improve sensitivity and more amplified signal when compared
staining and minimize false positive or negatives. to fluorochrome- or enzyme-conjugated secondary
• Secondary antibodies are used when there are no antibodies.
conjugated primary antibodies available or the primary • For best results, use secondary antibody that has been
antibody is not conjugated to a desired enzyme or preadsorbed with serum from the same species as
fluorochrome. the sample. This will reduce the background. However,
• Secondary antibodies are also used to increase these preadsorbed antibodies may have reduced
the sensitivity of detection. Even though use of a epitope recognition and may fail to recognize some
secondary antibody involves extra steps, it does IgG subclasses.
have the advantage of increased sensitivity due to • Affinity-purified secondary antibodies will provide
the signal amplification from multiple secondary the least amount of nonspecific binding. However,
antibodies binding to a single primary antibody. sometimes IgG fractions are preferred when it
• Secondary antibodies are generated by immunizing contains high affinity antibodies. This is of great
a host animal with the antibody from a different advantage when the antigen is present in very low
species. For example, anti-goat antibodies are raised levels.
by injecting goat antibodies into an animal other than • Select a secondary antibody that matches the class or
a goat. Accordingly, if the primary antibody is raised subclass of the primary antibody used. For example, if
in mouse, then secondary antibody should be an the primary antibody is mouse IgM then it is best to
anti-mouse antibody raised in another species (goat, use an anti-mouse IgM secondary.
donkey etc.) • When the class or subclass of the primary mouse
• For ELISA detection, enzyme-conjugated antibodies are monoclonal antibody is unknown, then anti-mouse
the better choice. For flow cytometry, it is best to use a IgG may be used, because it will recognize most of
fluorochrome-conjugated secondary antibody. mouse IgG subtypes.
• A vast majority of primary antibodies belong to the
Substrate
IgG class. They can be detected with the relevant
anti-species IgG secondary antibody. If the primary
antibody is an IgM, then the secondary antibody Conjugated Enzyme Detectable
specific for IgM should be selected. End Product

Secondary Antibody

Primary Antibody

Figure 6.
Principle of antigen detection using primary and secondary
antibodies

24
 ANTIBODY PRACTICE
2.6 Proper Controls of all dilutions, diluents, incubation times, lot numbers,
The use of proper controls will help eliminate any false preparation dates of all reagents, and procedural steps.
positive and false negative results and will enable better This information is highly valuable in efficient assay
interpretation of the experimental data. The key to development.
proving antibody specificity is often the correct use of
controls. They will also be invaluable in troubleshooting Isotype controls are used to validate that the primary
throughout the experimental design process. Here are antibody binding is specific and does not result from
some considerations: background signal due to immunoglobulins binding
nonspecifically. Typically, an isotype control is matched
• Whenever possible, both negative and positive controls to the host species and isotype of the specific primary
should be included in an assay. antibody. For example, IgG2a type antibodies raised
• A positive control sample may be any tissue, cell in mice can bind strongly to some human leukocytes.
line, or purified protein that is known to contain Hence, a mouse IgG2a isotype control should be used
the antigen of interest, and has been previously when analyzing human cells and tissues. Isotype controls
demonstrated to be positive by a reliable method. are most commonly used in immunoprecipitation,
• A negative control sample is one that is known to be flow cytometry, and immunohistochemistry. In many
devoid of the antigen of interest. A cell line or tissue flow cytometry applications, directly labeled primary
that is known not to express the protein of interest is a antibodies are used. Here, it is important to use
better negative control. the isotype control that is conjugated to the same
• In addition to sample controls, one should also use fluorochrome or label as the primary antibody.
reagent controls.
• Remember to change only one experimental variable
at a time.
Nice to know
• One should run separate controls for primary and Affinity maturation is an adaptive response to
secondary antibodies. antigen exposure. It is a process of affinity-
• Because antibodies from different animal bleeds or selected differentiation of activated B cells.
purification batches may have significantly different When the host animal is repeatedly exposed to
titer values, each new batch of antibody must be the same antigen it provokes greater antibody
validated, and conditions optimized before use in an ligating affinity. With the passage of time, the
existing assay. antibodies produced are able to bind antigen
more tightly and to deal more efficiently with
It should also be noted that an integral part of good the antigen.
laboratory practice is to keep complete documentation

2.7 Publishing with Antibodies of publications and grants are becoming increasingly
Let’s return to the basic immunolabeling assumption that critical of data analysis, and researchers are being
regardless of technique used, a positive signal infers that challenged to think about the fundamental principles
the specific antibody has bound to the specific antigen. by which laboratory techniques work, and to be more
As with any technique, it is good science to verify that careful about over-interpretation. The chart (on the next
your signal is indeed specific and reproducible. Reviewers page) should help bolster confidence in immunodata.

25
ANTIBODY PRACTICE

Actual reviewer comments Good practice

I am not convinced your Use two or more different techniques to verify specificity; for example,
antibody is specific WB can corroborate IHC data
Use two or more antibodies made against different immunogens or regions of the
protein and measure co-localization
Test against relevant knockout samples
I am not convinced your antibody Use two or more antibodies made against unconserved epitopes of the same antigen
is not cross-reacting with related to confirm results
proteins
Your Western blot shows more than Cite published literature on cleavage products or glycosylation patterns
one band or at the wrong size. How Consider running a denatured vs. native gel
can you show specificity?
Reprobe with a different antibody to same protein
Error bars are disturbingly large on Lock down your antibody protocol and then ensure you have enough antibody from
your antibody-based data the same lot number, so you don’t have to re-optimize each experiment because of
lot-to-lot variability
How much of the signal is actually Optimize protocol and reduce variability (see above).
background? Perform a peptide inhibition assay
Perform experiment without the primary antibody to establish background
Co-localize with direct fluorescent labeled primary
Use species preabsorbed secondary antibodies
Repeat your experiment with Many monoclonals are available for targets recognized by polyclonals
monoclonal antibodies for better data Choose a polyclonal made from a short peptide thus minimizing clonality and epitope
interpretation
Choose a polyclonal antibody validated in multiple applications to demonstrate
specificity across sample matrices, epitope treatments and detection environment
Redo experiment using antibody with Many antibody sequences are published by researchers or commercial suppliers and
known epitope. can be requested
Sequenced epitopes are not necessary for verifying antibody specificity or experiment
reproducibility
Publish antibody catalog number and company to aid in peer validation of your data

2.8 Validating Antibodies During validation, it must be shown that not only are
Validation is a process whereby, through the use of the antibodies specific and selective, but they can also
specific laboratory procedures, the performance and provide reproducible results. Hence, reproducibility is the
characteristics of an analytical technique are deemed final step in the validation process.
suitable for the intended use.
Antibody validation is particularly important in
Usually, the first test for antibody specificity is Western immunohistochemistry applications. Analysis of IHC data
blotting of a variety of cell line lysates with known levels can be challenging due to pre-analytical, analytical, and
of target expression. Here, both positive and negative post-analytical factors that affect staining, particularly
control cells are used. The next step in the validation with free-floating or paraffin-embedded sections. These
process is often immunohistochemistry (IHC) or factors are discussed in detail in section 3.7.
immunofluorescence (IF) to titer the antibody on tissue
samples.

26
Notes

27
28
 Antibody Applications
Antibody Applications 3
3.1 Introduction to Antibody Applications 3.6 Multiplexed Bead-based Detection
3.2 Immunoprecipitation 3.7 Immunohistochemistry/
3.3 Chromatin Immunoprecipitation (ChIP) Immunocytochemistry
3.4 Western Blotting 3.8 Flow Cytometry
3.5 Enzyme-linked Immunosorbent Assays 3.9 Functional Blocking and Stimulation Assays
(ELISA)

3.1 Introduction to Antibody Applications

The recognition of the value of the specific antibody-antigen interaction at the end of the 19th century has led to
the emergence of a variety of immunotechnologies, most of which are still in use today. From the precipitin test for
analyzing blood components to highly specialized chromatin immunoprecipitation techniques to immunostaining
in automated imaging flow cytometry, antibody-based tools continue to play an important role in biological and
biomedical research. In this section, the theory and practice of the major modern immunotechniques used today will
be discussed.

Timeline of Immunotools

1900. 1938. 1962. 1975. 1986.

Ehrlich Marrack proposes Edelman & Porter Kohler & Milstein Hepatitis B
offers Antibody antigen-antibody elucidate antibody develop monoclonal vaccine produced by
Formation Theory binding hypothesis. molecular structure. antibody production. genetic engineering.

1900. 1942. 1953. 1971. 1984.

Landsteiner & Coons et al. Grabar & Williams Perlman & Engvall Gilmor &
Levine use natural publish first IHC publish first invent ELISA. Lis describe ChIP.
antisera to recognize study. Antibody papers on
ABO blood groups. was labeled with immunelectrophoresis. 1965. 1980.
FITC. Fulwyler publishes Nadler et al.
1900. fluorescence-activated publish“Serotherapy”
Nuttall, Wasserman cell sorting paper. paper describing
& Schutze use 1960. lymphoma treatment
precipitin test to Sussman & with a targeted
distinguish human, Berson develop monoclonal antibody.
cow & goat milk 1946. RIA.
Elek, Oudin & 1979.
1900-01. Ouchterlony publish Renart, Reiser
Uhlenhuth work on on gel diffusion & Stark describe
egg-white typing technique for antigen- Western blotting.
paves way for use of antibody patterns.
precipitin reaction 1979.
in forensic work on Horan et al.
human blood stains develop bead-based
antibody multiplexing
assay.

29
Antibody Applications

3.2 Immunoprecipitation

3.2.1 Introduction
3.2.2 Antigen Labeling
3.2.3 Sample Preparation

Key Steps
3.2.4 Formation of the Antibody-Antigen
Sample in Solution
Complexes
3.2.5 Precipitation of Immune Complexes
3.2.6 Analysis
3.2.7 Troubleshooting

3.2.1 Introduction
It is possible to use antibody-antigen precipitation, or
immunoprecipitation (IP), to isolate a specific antigen
from complex protein mixtures in cell or tissue lysates.
Immunoprecipitation has proven to be an invaluable Add Antibody
investigational tool that is routinely employed by many
laboratories to ascertain critical information regarding
a given antigen. These include small-scale antigen
purification for functional studies, N-terminal sequence
analysis, investigation of protein-protein interactions,
and the determination of the relative abundance and
stoichiometric distribution of the antigen within a cell
or tissue. Success in an immunoprecipitation assay
is dependent on two main factors: the abundance of
antigen in the original sample and the affinity of the Add Protein A or
antibody for the antigen (normally requiring affinities of Precipitating 2° Ab
108 M-1 or higher).

Before beginning the immunoprecipitation procedure,

ensure that proper experimental controls are in place.
Control antibodies should be as similar in nature to the
specific antibody as possible. For a polyclonal antiserum, Centrifuge

the ideal antibody negative control would be the pre-

immune serum from the same animal. However, an equal
concentration of non-immune (normal) serum from a
different animal of the same species should suffice in its Discard Supernatant
absence.

Purity Complexes
Immunoprecipitation can be divided into the
following key steps:
IB Electrophoresis
• Antigen labeling (optional)
Enzymatic Studies
• Sample preparation (lysis of cells to release
the antigen) Ligand Binding
• Formation of the antibody-antigen (immune) complex
Immunoprecipitaion

• Precipitation of the immune complexes

• Analysis Figure 7.
These steps and troubleshooting will be discussed in
greater detail below.

30
 Antibody Applications
3.2.2 Antigen Labeling (optional) to recognize the target antigen. For a thorough review
Antigen may be labeled by incubating in a medium of the characteristics of some commercially available
containing a radioactive precursor (such as detergents, please see EMD Millipore’s Essential
H-thymidine), by iodination or biotinylation of
3 Biochemicals handbook (Available at:
surface proteins, by treatment with radioactive sodium www.emdmillipore.com/biochemicals).
borohydride, or by other published techniques. Antigen
labeling is optional, and is not required for most IP Regardless of the lysis strategy employed, the lysis buffer
assays. should always contain a relevant cocktail of protease
inhibitors (see Appendix for recipe). to protect against
3.2.3 Sample Preparation proteolysis of the target antigen by proteases liberated
Prior to formation of antigen-antibody complexes, the during the cell lysis procedure. Additionally, all solutions
antigen must first be efficiently extracted from the should be pre-chilled, and all steps in the lysis procedure
cell or tissue sample in a form that is still recognizable should be performed on ice.
by the antibody. This requires taking into account
the location of the antigen within the cell (nuclear, The number of cells required for lysis will vary with cell
cytosolic, membrane-bound, etc.) and determining how line and the anticipated abundance of the target antigen
best to extract the antigen with minimal effect on its in the sample. In general, lysates should be prepared
structural integrity. Before initiating an extraction/cell from no less than 107 cells at 107 cells per mL of lysis
lysis protocol, consider what information you hope to buffer. Following preparation of the lysate, determine
obtain from the immunoprecipitation. For example, is it the protein concentration and adjust concentration of
necessary to extract functional protein for subsequent the lysate to between 2 to 5 mg/mL with lysis buffer or
functional studies? Additionally, you may want to PBS. Extracts that will not be used immediately should be
consider whether to disrupt protein-protein interactions aliquoted, snap-frozen, and stored at -70°C for
or to co-immunoprecipitate proteins that may interact future use.
with the target antigen within the cell. Based on these
and other considerations, decide upon an appropriate
lysis strategy. Often, an effective lysis strategy is
determined empirically.
Watch Out
Perhaps the most important aspect of the lysis procedure If Triton® X-100 was used in the lysis
is the composition of the lysis buffer. The ionic strength buffer, the protein concentration cannot be
(salt concentration), choice of detergent, and pH of the determined by absorbance of the solution at
lysis buffer may significantly affect the efficiency of 280 nm as Triton® X-100 absorbs strongly at
extraction and integrity of the antigen. Slightly alkaline 280 nm.
pH and low ionic strength buffers typically favor protein
solubilization, while high salt concentration and low pH In general, direct protein quantitation via
may cause the antigen to denature and precipitate from an FTIR spectrometer, such as the Direct
solution. The choice of detergents is crucial and may be Detect® Quantitation System, enables accurate
influenced by many factors, including (among others) the measurements of total protein in many lysates,
subcellular location of the antigen, and whether the goal even in the presence of detergents, reducing
is to preserve subunit associations and other protein- agents and lipids.
protein interactions.

In general, non-ionic (e.g., Triton® X-100, NP40) or

zwitterionic (e.g., CHAPS) detergents tend to preserve
Tip
If the protein concentration of the extract is
noncovalent protein-protein interactions, while ionic
Immunoprecipitaion

below 0.1 mg/mL, high quality BSA should be

detergents (e.g., SDS, sodium deoxycholate) tend to
added to 1% (w/v) prior to freezing.
be more denaturing of protein-protein interactions,
and may adversely affect the ability of the antibody

31
Antibody Applications

3.2.4 Formation of the Antibody-Antigen for an additional 30 minutes at 4°C prior to adding
Complex Protein A/G). When Protein A or G Agarose is used for
Once the antigen has been extracted, antibodies are precipitation, 10–20 μL of a 50% Protein A/G agarose
added to the lysate to allow formation of the immune slurry should be sufficient to precipitate the quantity
complex. of antibody/antigen complex prepared according to
the procedure above. Following addition of Protein A/G
3.2.5 Precipitation of the Immune Agarose, incubate with gentle agitation for 30 minutes at
Complexes 4°C, then wash three times (or more, if the antigen has
To precipitate immune complexes, you may use:
been radiolabeled) by centrifugation and resuspension
• Protein A or Protein G Agarose,
in immunoprecipitation buffer, and collect antibody-
• Precipitating secondary antibodies, or
antigen-Protein A/G complex by centrifugation.
• Protein A-bearing S. aureus cells.

3.2.6 Analysis
The affinity of an antibody for Protein A or G is
Immunoprecipitated pellets to be used for electrophoresis
dependent on the subclass of the immunoglobulin and
may be resuspended with 2x SDS sample buffer and
the species from which it came. For example, Protein A is
resolved by SDS-PAGE (for subsequent detection by
exceptionally well-suited for immunoprecipitation of all
Western blot; please see section 3.4). The purified
rabbit primary antibodies, but not for chicken antibodies.
immune complex may also be used for enzymatic studies,
(See Protein A/G Binding Affinities in Appendix).
ligand binding, further immunizations, or for use in other
immunochemical techniques. These methods, when used
To use protein A for immunoprecipitation of mouse
in conjunction with immunoprecipitation, can greatly
primary antibodies, it is advisable to add 5 μg of rabbit
increase the amount of information about an antigen.
anti-mouse IgG (secondary precipitating antibody) prior
to the addition of Protein A/G (mix gently, and incubate

Technology Highlight

Faster, Simpler, More Reproducible IP

EMD Millipore’s Catch and Release® Equilibrate Columns Bind Wash Elute
The column contains a resin slurry that Within the column: Unbound Bound specific
kit was designed to meet your needs binds the antibody capture affnity • Specific protein binds Ab non-specific proteins are eluted
ligand (ACAL) • Ab binds ACAL proteins are
by making IP faster, simpler, and • ACAL binds resin removed Specific protein is in
• Complex is immobilized complex with Ab &
more reproducible. With in column ACAL
Catch and Release® kits, you can
purify both native and denatured
proteins using a variety of antibodies
• Remove snap-off • Add: • Wash to
and sample types. The kits are even bottom plug • Sample remove • Elute specific
• Drain buffer • ACAL unbound, protein of
validated for Co-IP and IP-kinase • Wash 2X • Ab non-specific interest
• Incubate proteins
assays.
Legend
Column Resin Specific Protein
Antibody Capture Affinity Ligand (ACAL) Non-specific Protein
Antibody (Ab)

Description Qty Cat. No.

Catch and Release® v2.0 Reversible Immunoprecipitation System 50 assays 17-500
Immunoprecipitaion

Catch and Release® v2.0 Reversible Immunoprecipitation System 5 assays 17-500A

Catch and Release® v2.0 High Throughput (HT) Immunoprecipitation Assay Kit- 96 well 96 assays 17-501
Catch and Release® Phosphotyrosine, clone 4G10® 50 assays 17-502
Catch and Release® Phosphotyrosine, clone 4G10® 5 assays 17-502A

32
 Antibody Applications
3.2.7 Troubleshooting
The most common challenge with immunoprecipitation is trying to lower the number and type of background proteins
that contaminate the washed immune complexes. Background problems can arise from many different sources that
may be either specific or nonspecific. Here are a few suggestions to deal with nonspecific background problems:

Problem Possible Cause Solution

High Background Contaminating proteins in Preclear the lysates with Protein A/G agarose beads prior to
the lysate adding the primary antibody.
Add saturating amounts of competitor proteins, such as BSA,
gelatin, acetone powders, or nonfat dry milk.
Spin the lysate at 100,000 x g for 30 min (discard pellet) prior
to addition of primary antibody.
Contaminants in antibody Centrifuge the antibody at 100,000 x g for 30 min
solution (discard pellet) and titrate.
Precipitating antibody is Try a different antibody.
nonspecific
Not enough wash time Increase the number of washes. “Soak” solid phase in the
wash buffers for 10 min per wash.
Too much antibody Decrease primary antibody concentration.
Incubations too long Decrease primary antibody incubation time.
Secondary antibody is If using rabbit anti-mouse immunoglobulin (precipitating
binding non-specifically secondary antibody) in conjunction with a monoclonal
antibody, check the background due to precipitating
secondary antibody alone. Titrate if necessary.
Protein A/G beads causing Run a control with only Protein A/G and without the primary
background antibody during Western blot analysis to determine if
background bands are from the Protein A/G beads or extract
alone.
Need to optimize Lower the number of counts per minute (cpm) of the
counting parameters (for radiolabel used to the minimum needed for antigen
radiometric assays) detection.
Multiple bands detected in Antigen may consist of Additional co-immunoprecipitated proteins are often useful
Western blot analysis more than one polypeptide in understanding antigen interactions, and must not be
chain or may have considered as a problem.
additional associated
polypeptides.
Coeluted capture Use crosslinking IP (covalently linking capture antibody to
antibody’s heavy or light Protein A/G) to avoid antibody coelution.
chain interferes with
target protein detection.
Multiple bands detected below May indicate proteolytic Prepare new lysates with fresh protease inhibitors added.
the anticipated molecular weight degradation of sample, Protease inhibitor cocktails may be used for this step.
possibly due to protease
inhibitor cocktail being old
Immunoprecipitaion

33
Antibody Applications

3.3 Chromatin Immunoprecipitation

(ChIP) Epigenetics Research
Epigenetics describes heritable changes in gene
3.3.1 Introduction
expression caused by non-genetic mechanisms
• Sample Preparation*
• Crosslinking Proteins to DNA* instead of by alterations in DNA sequence.
• Cell Lysis and Chromatin Fragmentation* These changes can be cell- or tissue-specific,

Key Steps
3.3.2 Chromatin Immunoprecipitation and can be passed on to multiple generations.
• Washing, Elution, and Crosslink reversal* Epigenetic regulation enriches DNA based
• DNA Purification and Cleanup* information, allowing a cell to vary its response
• PCR Analysis*
across diverse biological and environmental
3.3.3 Advances in ChIP Technology
3.3.4 Troubleshooting contexts. Epigenetic changes can effect
3.3.5 FAQs transcriptional and post-transcriptional
regulation via the following mechanisms:

3.3.1 Introduction • Histone modifications

Chromatin immunoprecipitation (ChIP) is a powerful • Positioning of histone variants
technique classically used for mapping the in vivo • Chromatin and nucleosome remodeling
distribution of proteins associated with chromosomal • DNA methylation
DNA. These proteins can be histone subunits, • Small and non-coding RNA-mediated
transcription factors, or other regulatory or structural epigenetic regulation
proteins bound either directly or indirectly to DNA. These mechanisms, in cooperation with
transcription factors and other nucleic acid-
Successful ChIP requires high quality ChIP-validated binding proteins, regulate gene expression,
antibodies that can specifically detect proteins associated resulting in cellular diversity although DNA
with target regions of chromosomal DNA. Traditionally, sequences are virtually identical from cell to
endpoint and/or quantitative PCR (qPCR) are performed cell. Epigenetic mechanisms of gene regulation
after ChIP to verify whether a particular DNA sequence impacts diverse areas of research—from
is associated with the protein of interest. Using this agriculture to human health.
classical approach, researchers can evaluate the
interactions of the proteins of interest with a limited
number of known target genes.

The use of antibodies in chromatin immunoprecipitation

The ChIP experiment can be divided into the
is discussed in greater detail in the following pages.
following main steps:
• Sample preparation*
* Resource:
• Crosslinking proteins to DNA*
For a thorough discussion of key steps in ChIP,
• Cell lysis and chromatin fragmentation*
refer to EMD Millipore’s Guide to Chromatin
• Chromatin Immunoprecipitation
Immunoprecipitation: Critical Factors for Success
• Washing, elution, and crosslink reversal*
(Literature Number TP5994EN00).
• DNA purification or cleanup*
• PCR analysis*
Immunoprecipitaion

34
 Antibody Applications
Cells Tissues
ChIP kits make chromatin
In vivo cross-linking immunoprecipitation a snap!
Lysis
Chromatin Immunoprecipitation (ChIP) assays
Isolation of chromatin
cultured cells or tissues have become very easy with the availability
of assay kits that contain reagents optimized
for immunoprecipitation of transcriptionally
Sonication to shear
chromatin active chromatin from mammalian cells. The
detection of the gene or promoter of interest
in immunoprecipitated chromatin must still
be empirically determined by the researcher.
Various detection techniques can be used:
96-well plate either quantitative PCR, sequencing, or
immunoprecipitation
• 10,000-100,000 cells Southern slot-blot analysis is recommended.
per reaction
• Manual or automated A Chromatin Immunoprecipitation (ChIP) Kit
processing
may include the following reagents:

• Control DNA
• Buffers for dilution, wash, incubation etc.

Including: Low salt, High salt, LiCl, Tris-EDTA,

EDTA, NaCl, Tris-HCl, SDS Lysis Buffers
Reversal of cross-links
In addition, an antibody specific to the target
of interest is required, such as an Anti-acetyl
Histone H3 or H4.

DNA purification (optional)

Amplification
2000

1500 Detection
• Quantitative PCR
RFU

1000

500
• Promoter microarray
0
• Sequencing
0 10 20 30 40 50
Cycles

Figure 8.
Chromatin Immunoprecipitation Workflow. Immunoprecipitaion

35
Antibody Applications

3.3.2 Chromatin Immunoprecipitation Regardless of your choice of either monoclonal or

The goal of this step is to use appropriate antibodies to polyclonal, when selecting a commercially prepared
isolate target protein/DNA complexes from chromatin antibody for ChIP, the ideal antibody will have data
extracts prepared in previous steps. demonstrating specificity as well data showing reliable
performance in ChIP and other key applications.
Selecting an appropriate ChIP antibody is the one of the
most critical steps toward a successful ChIP experiment. Antibodies that are in high demand from commercial
Even the highest quality antibodies, which may perform suppliers often need to be remanufactured, starting with
very well in typical Western blot validations, may not be the immunization of a host animal. Consequently, the
suitable for ChIP. It is best to consider only antibodies specificity and affinity of these antibodies can vary from
that have been validated specifically in ChIP. If your batch to batch. Larger manufacturers of antibodies such
antibodies are not specifically quality controlled and as EMD Millipore are able to address this by immunizing
proven to perform in ChIP (e.g. ChIPAb+™ validated multiple animals followed by screening and pooling
antibody primer sets) we suggest you evaluate several of materials demonstrating appropriate affinity and
potential antibodies before selecting one for your specificity. To ensure consistency, the performance of the
actual ChIP experiments. Below are a few parameters to final antibody can be compared to previous batches.
consider before selecting your antibody.

Monoclonal vs. Polyclonal Antibodies Tech Tips

Either monoclonal or polyclonal antibodies will work • Whether you select a monoclonal or polyclonal
for ChIP. A monoclonal antibody recognizes a specific antibody for your ChIP experiment, you must
epitope on the target protein. Monoclonal antibodies optimize the dilution of your antibody for your
specific analysis. If you use excess antibody,
provide the advantage of being highly specific with less
you may succeed in immunoprecipitating your
of a propensity for nonspecific binding. In addition, they
target protein, but you may also observe higher
perform more consistently from batch to batch due low nonspecific binding or reduced specific signal.
variability in their clonal nature. However, if the epitope In contrast, if use you use too little antibody
recognized by the monoclonal is masked or altered by you will typically observe low recovery of your
previous steps in the protocol, such as crosslinking, then target.

monoclonal antibodies will not be effective in isolating • For the best results, ChIP antibodies should be
your target protein and its associated DNA sequences. well characterized, proven to bind to its target
Fortunately, this masking rarely affects monoclonal protein, rigorously tested for specificity, and
ideally validated in ChIP.
antibodies.
• Just because an antibody works well in a
In contrast, polyclonal antibodies recognize multiple Western blot does not always indicate it will
epitopes of a target protein. A polyclonal antibody may perform well in chromatin immunoprecipitation.
Unlike a Western blot that detects proteins that
therefore be more effective even if a few epitopes are
have been denatured, a ChIP antibody must
masked by crosslinking. However, because polyclonals recognize the target protein in its native state.
recognize multiple epitopes, this can increase the
probability that nonspecific binding will occur. In
addition, it is important to also consider that the
specificity of the polyclonal population may drift over
time during immunization, unless the serum from which
the antibody is purified is pooled prior to preparation
or purification. A related point is that most commercial
polyclonal antibodies may differ from batch to batch. The
degree of variation will depend upon the manufacturing
Immunoprecipitaion

and quality control practices of the vendor. For example,

polyclonal antibodies to modifications have finite
amounts of serum available.

36
 Antibody Applications
Control Antibodies for ChIP is a simple Western blot-like procedure that works with
Chromatin is a complex mixture of proteins bound to standard chemiluminescent detection systems and
nucleic acid. To ensure valid results, ChIP experiments in contrast to microarray-based approaches, requires
should include controls for nonspecific binding of no additional software or expensive equipment for
chromatin to your antibody, immunoprecipitation beads, detection.
and the resultant ChIP’d DNAs to be analyzed. To control
for specificity of your ChIP antibody, you should include Validation Testing
the following controls in your ChIP experiment: Ideally an antibody will be validated in ChIP. However,
the lack of ChIP data is not always an indication that
• Negative control antibody: Use a negative control IgG an antibody will not work for ChIP. When selecting an
from the same species and format (e.g. purified, naive antibody for ChIP studies, consider all application data
serum, or ascites) as your ChIP antibody. For example, available for that antibody. Typically the greater the
if you use a normal, purified mouse monoclonal ChIP variety of applications for which there is data, the higher
antibody, use a normal purified mouse IgG as your the likelihood of an antibody performing well in ChIP. For
negative control. If you are using a purified rabbit example, an antibody validated in immunoprecipitation
polyclonal ChIP antibody, then use a normal, purified (IP), immunofluorescence (IF), or immunohistochemistry
rabbit IgG as a negative control antibody. Alternatively, (IHC) is more likely to produce positive ChIP results than
if an appropriate negative control IgG is not available, an antibody validated for only Western blot. However,
you may opt to use the “no antibody” or bead-only validation in these applications does not guarantee
approach. Although the matched IgG is typically a the success of the antibody in ChIP, as successful ChIP
better negative control, either the negative control IgG antibodies must recognize accessible epitopes that are
or a bead only control can be used for fold enrichment not affected by the crosslinking methods often used in
calculations. ChIP.
• Positive control antibody: Use an antibody of the
same species that recognizes an abundant protein
target, such as RNA polymerase II or histone subunits
Tech Tip
(typically H3 or H4 N-term). The positive control Select candidate ChIP antibodies that perform
antibody helps to validate aspects of the experiment in immunocytochemistry, Western blot, and/or
immunoprecipitation. Satisfactory performance
not affected by selection of your ChIP antibody and
in these other immunoassays increases the
can be helpful for troubleshooting experiments.
chances of performance in ChIP. Although this
does not guarantee performance in ChIP, it can
Specificity Testing
help prioritize candidate antibodies and screen
Before you select an antibody, it is important to out less desirable ones.
understand the specificity of your antibody. This is
particularly true when working with antibodies directed
against post-translationally modified proteins, such as Application of ChIP Antibodies
modified histones. It is important to confirm that the The ChIP antibody may be conjugated directly to agarose
antibodies used detect only epitopes containing your or magnetic beads or it may be immobilized on beads
specific modification site. There are multiple approaches conjugated to protein A, protein G or a protein A/G bead
to evaluate the specificity of an antibody, including: dot blend. Original ChIP protocols used agarose beads, but
blots, peptide microarrays; peptide microarrays or peptide many long-time ChIP users have moved to magnetic
inhibition assays. Some labs will perform a number beads. Magnetic beads enable rapid isolation of protein/
of validation methods, especially for modified histone DNA complexes from the crude chromatin mixture using
peptides, to properly evaluate cross-reactivity to similar a magnetic separation device.
epitopes, for example between dimethyl and trimethyl
adducts on the same histone subunit. For a reliable Agarose beads perform well in the hands of many
Immunoprecipitaion

method of testing specificity for histone antibodies, we researchers and offer a less expensive, but more time-
recommend AbSurance™ Histone Antibody Specificity consuming option. These beads require centrifugation for
Arrays (Cat. Nos. 16-665, 16-667, and16-668). These separation and may exhibit nonspecific binding, which
arrays provide a total of 89 high quality peptides of may require blocking and lysate preclearing.
histones H2, H3, and H4 on PVDF membranes. The assay
37
Antibody Applications

The choice of bead type is often influenced by the Regardless of your choice of beads, the order in which
type of antibody one plans to use for the experiment. you apply magnetic or agarose beads to your ChIP
Protein A beads exhibit the highest affinity for rabbit reaction may affect your ChIP signal. One method is to
polyclonal antibodies, whereas the protein G beads bind incubate the beads with the capture antibody (few hours
a wider range of antibodies including most, but not at room temperature, or overnight at 4°C) followed by
all, classes of mouse monoclonal IgGs. The protein A/G addition of chromatin and further incubation (1 hour
blend provides the most flexibility in terms of the type to overnight with rotation, at 4°C). Increasing the time
of antibody that you can use, because it combines the of incubation may increase both the background and
binding characteristics of both protein A and protein the ChIP signal; however, antibodies with low affinities
G. Our research and development team has found for their targets generally do not produce significant
that the protein A/G blend typically produces lower ChIP signals without longer (overnight) incubations.
backgrounds than protein A or G alone, for a wide range Alternatively, some protocols involve either incubating
of antibodies without compromising the efficiency of the antibody with chromatin and then adding beads or
immunoprecipitation. adding all three components at the same time. Adding
all three components often works, and reduces the time
required to perform the overall reaction.
Magnetic Beads
Advantages 3.3.3 Advances in ChIP Technology
Low nonspecific binding Epigenetic marks are as important as DNA sequences in
Blocking and preclearing not required determining phenotypes and their study has heightened
Easy to handle during washes focus on protein-DNA interactions versus traditional
Beads are visible in tube gene-based heredity studies. Characterization of
Reproducible results these epigenetic patterns and regulatory networks
Disadvantages has progressed beyond standard chromatin
Slightly higher cost immunoprecipitation (ChIP) based techniques to include
Magnetic rack required ChIP-chip, genome-wide microarray analysis of ChIP-
Non-porous (binding capacity dependent isolated DNA, and ChIP-seq (ChIP followed by sequencing
on bead surface area)
of the immunoprecipitated DNA).

Agarose Beads These high content approaches provide insights into how
regulatory and structural proteins, such as transcription
Advantages
factors and histone subunits, bind and interact with the
High capacity binding (porous), which may also
increase nonspecific binding genome. Immunoprecipitation of these protein-DNA
Simple equipment required complexes through high quality antibodies selective
(centrifugation or filtration) against post-translational modifications, combined with
Slightly lower cost sequencing or microarrays, identify sequence-specific

Disadvantages DNA binding sites with precise resolution.

Blocking required
Preclearing required
Not visible in tubes
High probability of bead loss during handling
Immunoprecipitaion

38
 Antibody Applications
3.3.4 Troubleshooting:
Problem Possible Cause Suggested Steps
High background in negative Excessive antibody Optimize the concentration of the antibody.
control (IgG or mock IP) samples resulting in binding to
non-targets:
Nonspecific binding to Include a pre-clearing step to exclude these non-targets or add a
beads: blocking agent to the beads.
Incomplete fragmentation Optimize the fragmentation process to acquire chromatin lengths
of chromatin: between 200-1000 bp. Separately optimize fragmentation for each cell
or tissue type. Use siliconized or low retention tubes.
Contaminated reagents: Ensure that all reagents are freshly prepared and free of contaminants.
Increase the number of washes.
Run a “no DNA” PCR reaction to determine if your sample is
contaminated with nucleic acids.
“No DNA” PCR reaction is Use pipettes dedicated to PCR, and UV-irradiate pipettes prior to setting
showing signal up PCR.
Perform ChIP, DNA purification, and PCR reaction setup in three separate
rooms/areas using dedicated pipettes, or set up reactions in a hood.
Avoid using bottled or otherwise prepackaged water. Use freshly
delivered Milli-QR water from a system containing a UV light source.
Use aerosol-resistant pipette tips.
Do not open tubes containing amplified PCR products anywhere near
the location of future ChIP or qPCR experiments.
Low recovery of DNA Ineffective or low affinity Ensure that you are using an antibody that has been validated in ChIP
ChIP antibody: For a complete selection of ChIP antibodies, see www.emdmillipore.com/
epigenetics. If you are using a ChIP antibody, increase the incubation
time of the antibody.
Insufficient ChIP antibody: Generally 1-10 μg of ChIP antibody is sufficient. However, the amount
of antibody required may depend on the relative abundance of your
target protein and the affinity of the antibody for the target.
Insufficient starting Before crosslinking, prepare a separate plate to determine cell number.
sample: Re-evaluate your cell number per reaction. Increase your cell number
especially if you are attempting to detect a low abundance target.
Incomplete cell lysis and Optimize these steps by varying parameters (see section 5.3) and
ineffective fragmentation: assessing their effects on chromatin recovery. Use mechanical force
such as a dounce homogenizer or glass beads to improve cell lysis.
Optimize the fragmentation steps and avoid foaming.
Over-crosslinking: Long incubation in formaldehyde may mask epitopes required for
recognition by ChIP antibody. This can be especially problematic if you
are using a monoclonal antibody. Over crosslinking may also result in
the formation of complexes that are resistant to sonication. Optimize
crosslinking steps: the final concentration of formaldehyde should be
1%, and you should determine the most effective crosslinking time
before proceeding with the experiment.
Under-crosslinking: Insufficient crosslinking may result in dissociation of target proteins
from DNA during subsequent steps of the protocol. Unless you are
studying histones and histone modifications, you should use an X-ChIP
protocol to stabilize the associations of your target protein with DNA.
Increase crosslinking time.
Low affinity or low quality Protein G magnetic beads bind a wider range of antibodies, including
beads: mouse monoclonals. For the most flexibility with antibody choice, we
recommend the protein A/G blend (Cat. No. 16-663).
PCR primers: Test the efficiency of the primers. Include appropriate controls and
redesign primers if necessary.
Immunoprecipitaion

For a more thorough Troubleshooting guide, refer to EMD Millipore’s Guide to Chromatin Immunoprecipitation:
Critical Factors for Success (Literature Number TP5994EN00).

39
Antibody Applications

3.3.5 Frequently asked questions:

Question Answer
Should I use a Either monoclonal or polyclonal antibodies can work for ChIP. Monoclonals are frequently
monoclonal or highly specific, but monoclonals can be sensitive to crosslinking conditions. Over crosslinking
polyclonal antibody? may mask the target epitope and careful optimization of crosslinking might be required.
Polyclonals are less sensitive to over crosslinking conditions, and may produce better
enrichment than comparable monoclonals, but polyclonals are more likely to bind to
nonspecific targets.
How can I increase the Test specificity/crossreactivity to identify the epitopes recognized by your antibody.
chances of the selected You will also need to test the antibody in multiple immunoassays such as Western blot,
antibody working in immunocytochemistry, and immunoprecipitation, then make sure the antibody produces
ChIP? good fold enrichment of your target DNA in ChIP.
How much antibody We recommend using 2-10 μg of your ChIP antibody depending on the abundance of your
should I use? protein target and the affinity of your antibody for the target. More antibody does not always
equal stronger signal. It is suggested that to get the best ChIP signal the amount of antibody
be titrated.
How should I choose Choose an antibody that has passed multiple specificity/crossreactivity tests, and validated in
a commercial ChIP ChIP and multiple immunoassays. For screening of your histone antibodies, we recommend
antibody? the AbSurance™ histone antibody specificity Arrays (Cat. Nos. 16-665, 16-667, and 16-668).
Do you recommend Using a tagged antibody in ChIP is a way to get around antibody unavailability, variability
using tags if I cannot and epitope masking in crosslinked chromatin. It is possible that a tag will interfere with
find a suitable ChIP transcription factor function. Tags should be evaluated on a case-by-case basis. Switching
antibody for my study? tags between N and C termini may be good controls.
What is a good control We recommend using normal IgG from the same species as your ChIP antibody, so if you are
antibody? using a mouse monoclonal, we recommend normal mouse IgG.
What is the advantage Many antibodies bind to both protein A and G with varying affinity and specificity. Blending
of using protein A/G protein A and G beads eliminates the need to choose one over the other and to evaluate
bead blend? binding to both types for optimization. In most cases we have seen better fold enrichment
and reduced background activity using a protein A/G bead blend compared to using similar
quantities of pure protein A or protein G beads.
What is an acceptable The IgG pulldown can be quite variable and qPCR-assay dependent. The same mock IgG
% input range for the sample can have different percent input results in one location of the genome vs. another
normal IgG control region based on sequence composition of the assay design. The signal may be the result of
antibody? nonspecific binding of nucleic acid to tube, to beads, to antibodies. ChIP is relative so it is
best not to attempt to conform to a specific percent of input value, but ideally, the IgG value
should have Ct values that are nearest to the most dilute sample in your standard curve.
Again, ChIP is relative so if your ChIP signal is higher than your IgG signal (within limits of
variation in the assay), you have a positive ChIP result.
Immunoprecipitaion

40
 Antibody Applications
Technology Highlight

Reliable, Hassle-Free Results

Chromatin Immunoprecipitation Kits:
ChIP kits offer a ready-to-use and reliable approach to ChIP. At EMD Millipore we offer a variety of ChIP kits based on both magnetic
beads as well as agarose beads. Magnetic beads utilize a magnetic separation device for processing, and are generally preferred by
laboratories performing ChIP due to their ease of use along with better and more reliable recovery of input beads.

Magna ChIP® Kits Offer

• Full set of optimized and quality controlled reagents proven to work in ChIP
• Detailed protocols for cells and tissues
• Time savings: avoid making reagents and conducting multiple validation and troubleshooting experiments
• Faster protocols that enable ChIP in one day using magnetic beads
• Advanced protocols that enable up to 96 ChIP reactions at once in a single plate
• Positive and negative controls; EZ-Magna ChIP™ and EZ-ChIP™ kits come with IgG controls and well-designed qPCR primers
• Genome-wide kits for microarray and ChIP-seq analyses
• Expert technical support and troubleshooting

Description Cat. No.

Magna ChIP® A/G Kit 17-10085
EZ-Magna ChIP™ A/G Kit 17-10086
Magna ChIP® HT96 Kit 17-10077
EZ-Magna ChIP™ HT96 Kit 17-10078
Magna ChIP-Seq™ Chromatin Immunoprecipitation and Next Generation Sequencing Library Preparation Kit 17-1010
Magna ChIP2™ Universal Chromatin Immunoprecipitation DNA Microarray Quad Kit 17-1004
Magna ChIP® G Tissue Kit 17-20000
ChIP Assay Kit (Agarose) 17-295
EZ-ChIP™ Kit (Agarose) 17-371

Immunoprecipitaion

41
Antibody Applications

3.4 Western Blotting The basic Western blotting procedure involves the
following key steps:
3.4.1 Introduction • Sample Preparation
3.4.2 Sample Preparation • Gel Electrophoresis
3.4.3 Gel Electrophoresis
• Membrane Transfer

Key Steps
3.4.4 Membrane Transfer
• Blocking Nonspecific Binding
3.4.5 Blocking Nonspecific Binding
3.4.6 Addition of the Antibody • Addition of the Antibody
3.4.7 Detection • Detection
3.4.8 Common errors
3.4.9 Troubleshooting The guidelines below describe a starting point from
3.4.10 A Note on Membrane Selection
which you can develop your optimized processes.

3.4.1 Introduction
Resource:
Western blotting (WB) combines the resolution of gel
For a thorough guide to Western blotting success,
electrophoresis with the specificity of antibody detection.
troubleshooting and description of recent enhancements
Blotting can be used to ascertain a number of important
to the Western blotting techniques, refer to EMD
characteristics of protein antigens, including detecting
Millipore’s popular Protein Blotting Handbook
the presence and quantity of an antigen, the molecular
(Literature Number TP001EN00).
weight of the antigen, and the efficiency of antigen
extraction. This method is especially helpful when dealing
3.4.2 Sample Preparation
with antigens that are insoluble, difficult to label, or are
An unlabeled solution of proteins, frequently an extract
easily degraded, and thus not amenable to procedures
of cells or tissues is first prepared in a gel electrophoresis
such as immunoprecipitation.
sample buffer (see Useful Formulations section in the
Appendix).
By taking advantage of distinct physical characteristics
of different polypeptide species such as size, electrical
In some cases, the sample to be blotted may have
charge, and shape, a complex mixture of proteins can
been derived from immunoprecipitation, as described
be resolved chromatographically (electrophoretically)
previously. Please see 3.2.3 Sample Preparation in the
by applying the sample to a gel matrix in the presence
Immunoprecipitation section for more details.
of an electric current. The common technique used to
separate proteins in this manner is SDS-PAGE. A great
deal can be learned about the properties of a protein by
“running gels,” however, even more can be learned by Watch Out
transferring the fractionated protein sample from the gel Samples should not be boiled as proteins
to solid support membranes (blotting), and detection containing significant stretches of hydrophobic
with specific antibodies. Western blotting has become amino acids (such as membrane proteins) tend
very common procedure in life science research. The use to aggregate when boiled.
of quality antibodies in the detection of proteins on a
Western blot is critical to success.
3.4.3 Gel Electrophoresis
A charged protein migrates in an electric field relative
Western blotting remains the platform of choice for
to its net charge. However, as the molecule migrates
exploratory research, and is still the standard by which
through the gel matrix in response to the electric current,
new antibodies and other protein detection assays
its mobility is retarded depending on its size and shape
(such as ELISA, bead-based assays, flow cytometry and
by the sieving effect of the gel matrix.
immunohistochemistry) are evaluated. The development
of new technologies has yielded tools to improve signal-
Polyacrylamide gel electrophoresis (PAGE) can be used
to-noise ratios in Western blotting, and has greatly
Western blotting

to separate individual proteins by their size, shape,

reduced the time required for the Western blotting
and charge under non-denaturing conditions. This is
process (for example, EMD Millipore’s SNAP i.d.® 2.0
commonly called native PAGE. Under non-denaturing
system, Cat. No. SNAP2MM).

42
 Antibody Applications
conditions, the migration of some proteins is affected
by the retention of secondary and higher order structure Tech Tips
stabilized by covalent disulfide bonds between adjacent
• Make sure that the gel acrylamide
cysteine residues.
concentration is appropriate for the
anticipated molecular weight of the antigen
PAGE can also be performed under denaturing to be detected and that the acrylamide
conditions, typically in the presence of a molar excess of solution is degassed prior to casting gel.
the ionic detergent Sodium Dodecyl Sulfate (SDS). This is • If casting gels manually, always cast
commonly known as SDS-PAGE. SDS-PAGE gels the day before use to insure
complete polymerization for maximum
Furthermore, PAGE can be performed under denaturing resolution.
and reducing conditions (SDS-PAGE in the presence of a • Fresh ammonium persulfate and
reducing agent such as dithiothreitol (DTT) or tetramethylethylenediamine (TEMED) should
β−mercaptoethanol). be used to catalyze gel polymerization.
• Rinse wells of the gel thoroughly before
Denaturing conditions: applying sample.
Most often, polyacrylamide gel electrophoresis is
• Apply 10–50 μg of total cell or tissue lysates
performed in the presence of SDS. Prior to resolving
or 0.1–1.0 μg of a purified protein in 1x SDS-
the sample by SDS-PAGE, the protein is denatured by PAGE Sample Buffer per well.
heating the sample in the presence of the detergent.
• If samples are to be run under native, or
By disrupting non-covalent intra- and intermolecular
non-reducing conditions, β−mercaptoethanol
associations, the protein is effectively loses its secondary
and DTT should be excluded from the sample
and tertiary structure. As a consequence, the denatured buffer.
protein molecules become uniformly “coated” with
the negatively charged SDS at a concentration of
approximately 1.2 grams SDS per gram of protein,
thus giving the protein molecules a net unit negative Watch Out
charge per unit mass. Protein samples fractionated by Pre-stained molecular weight markers often
denaturing SDS-PAGE are, therefore, resolved roughly do not run true to size. It is recommended that
according to their relative molecular weight regardless of unstained molecular weight standards be run as
charge (and to some degree, shape). well for an accurate determination of antigen
molecular weight.
Reducing conditions:
Often, polypeptides containing intact disulfide linkages
migrate anomalously by SDS-PAGE. The resolution Tech Tips
of such proteins by SDS-PAGE is influenced by their • Samples should be heated at 50–65°C for
charge as well as their shape. This is due, in part, to 10–15 minutes prior to loading gel.
steric hindrance of SDS binding to the protein in regions • Run pre-stained molecular weight markers in
participating in the formation of inter- or intramolecular one well in order to monitor the transfer of
disulfide bonds, resulting in a heterogeneous charge protein from the gel to solid supports during
distribution across the molecule. Additionally, the the membrane transfer step. This will also
secondary structure stabilized by the disulfide linkages help to orient the gel during the transfer
affects migration through the gel matrix. To alleviate procedure.
this potential problem, a reducing agent such as DTT or • Following the specifications of the equipment
β−mercaptoethanol is added to the SDS sample buffer manufacturer, electrophorese the sample
to disrupt the disulfide bonds. Under reducing and through the polyacrylamide gel to resolve
denaturing conditions, all proteins in the sample should the protein by molecular weight. Stop
Western blotting

be resolved by SDS-PAGE according to size (molecular electrophoresis when the bromophenol blue
weight) alone. For this reason, SDS-PAGE is most dye front reaches the bottom of the gel.
commonly run under reducing conditions.

43
Antibody Applications

3.4.4 Membrane Transfer heavily coated with SDS when they leave the gel and
Proteins resolved by SDS-PAGE are transferred encounter the membrane, thus reducing the efficiency of
from the gel to a solid support membrane. This can protein binding to the membrane.
be accomplished by either capillary blotting or by
electroblotting (semi-dry and tank transfer systems). Conversely, higher molecular weight antigens typically
require longer transfer times.
The more efficient and most widely used method of
transfer is electroblotting. In this procedure, a sandwich Typically, the more hydrophobic a protein is, the
of gel and solid support membrane (nitrocellulose or more difficult it may be to transfer to solid support
polyvinylidene difluoride (PVDF)) is compressed in a membranes.
cassette and immersed in buffer between two parallel
electrodes. A current is passed at right angles to the gel, To transfer a protein from a gel to a membrane:
which causes the separated proteins to electrophorese Following SDS-PAGE, the gel is prepared for
out of the gel and onto the solid support membrane. electroblotting using a standard tank transfer or semi-
Once the proteins have been transferred to the solid dry blotting system. Generally, a transfer “sandwich” is
support membrane, the membrane is referred to as a assembled, with the following layers in order:
“blot”.
cathode (-) end
The efficiency with which a particular antigen will be 1) sponge or foam pad
transferred to the membrane is dependent on the protein 2) filter paper (3 sheets) soaked in transfer buffer
binding capacity of the membrane used, the transfer 3) gel with resolved proteins
method and conditions employed, as well as the nature 4) membrane (nitrocellulose or PVDF)
of the antigen itself. To maximize transfer efficiency, 5) filter paper (3 sheets) soaked in transfer buffer
some knowledge of the physical properties of the target 6) sponge or foam pad
antigen is beneficial. With respect to the efficiency of anode (+) end
transfer, the most important properties are size (MW)
and hydrophobicity of the antigen. While SDS is required The sandwich is assembled, and placed in the transfer
to facilitate migration of the protein out of the gel in system. The transfer is accomplished by applying 1
response to an electric current, SDS can also interfere ampere (constant current) for 1 hour, or equivalent, in
with binding of the protein to the membrane itself (this a wet transfer system, or at 0.7 amperes for 45 minutes
is particularly an issue with PVDF membranes). Since in a semi-dry transfer system, with 25 mM Tris, 190 mM
smaller polypeptides migrate faster, they may still be glycine, and 20% methanol (optional) as transfer buffer.

Western Blotting
(-) Cathode

Foam pad

Filter paper (x3)

Direction
Gel
of transfer
Membrane

Filter paper (x3)

Foam pad Block Membrane

Transferred Protein
(+) Anode Add 1° Ab
MW Markers
Add 2° Ab

Add Subtrate

E
Western blotting

Figure 9.
Detect Signal
Proper orientation of Western blotting
sandwich and sequence of reagent addition.

44
 Antibody Applications
Typical blocking solutions include:
Tech Tips • 10% (w/v) bovine serum albumin (BSA)
• 5% non-fat dried milk in Tris or
• Cut the membrane and filter paper (6 sheets)
phosphate-buffered saline
to fit the gel exactly.
• Protein-free blockers, such as Bløk™ reagents, which
• It is important that gloves are worn at all may improve signal-to-noise ratios by minimizing
times while handling the membrane to background signal
prevent contamination.
• Filter paper soaked in transfer buffer can be Incubation in blocking solution for 30 minutes at 37°C
used to carefully remove the gel from the or 1 hour at room temperature is sufficient to block
glass plates or plastic cassette, and to then membrane.
transfer the gel to the membrane.
3.4.6 Addition of the Antibody
• Remove all air bubbles between the gel and
Dilute the primary antibody in Tris- or phosphate-
the membrane. This can be done easily by
buffered saline. Unless nonspecific reactivity is observed
rolling a test-tube or Pasteur pipette across
or anticipated, it is not necessary to add blocking protein
the surface of the gel/membrane sandwich.
to the primary antibody. After decanting the blocking
• For longer transfer times, it is recommended buffer from the blot, incubate the membrane with
that electroblotting be performed at diluted primary antibody for 30 minutes at 37°C, one
4°C to prevent overheating and buffer hour at room temperature, or overnight at 4°C, with
decomposition. gentle agitation. Consult individual product datasheets
Note: To ensure complete transfer, the blot can be for suggested dilution ranges. Following incubation in
stained with Ponceau S without interfering with primary antibody, the blot is washed in several changes
subsequent immunodetection. The more sensitive
of wash buffer (Tris- or phosphate-buffered saline with
Coomassie® brilliant blue or amido black dyes can
be used to visualize protein bands, although, these 0.1% Tween® 20) before addition of secondary antibody.
reagents may be incompatible with subsequent Follow by incubation with a labeled secondary antibody
immunodetection. (as above).

3.4.7 Detection
3.4.5 Blocking Nonspecific Binding The method of detection is dependent upon the label
After the proteins have been transferred to a membrane, that has been conjugated to the primary (or secondary)
but before they can be visualized using antibodies and a antibody.
detection method, the membrane must be incubated with
a suitable blocking protein solution to block remaining The most common antibody label used in Western
hydrophobic binding sites on the membrane. This will blotting is an enzyme such as alkaline phosphatase or
reduce background and prevent binding of the primary horseradish peroxidase, which catalyzes either a light-
antibody to the membrane itself. producing reaction using a chemiluminescent substrate
or a color-producing reaction using a colorimetric
substrate (chromogen). Chemiluminescent signals can
Watch Out be detected by exposing the blot to X-ray film, while
colorimetric signals are detected visually.
It is important to be certain that the blocking
solution does not contain antigens that may
Fluorescent detection employs either a fluorophore-
be recognized by the primary or secondary
conjugated antibody or fluorogenic substrates
antibody. For instance, if using anti-phospho
that fluoresce at the site of enzyme activity
antibodies, it is recommended that Tris- (chemifluoresence). One advantage of this method is that
buffered saline (TBS) be used instead of the fluorescent signal is stable for long periods of time,
phosphate-buffered saline ( PBS), as the and blots can be archived and re-imaged. In addition,
latter may show nonspecific reaction to the the wide variety of fluorophores makes it possible to
antibody. Using a protein-free blocking reagent simultaneously detect multiple protein targets in a single
Western blotting

specifically designed for phosphospecific sample (multiplex detection).

detection (e.g. Cat. No. WBAVDP001) is
likely to minimize background signals during Some antibody detection systems, such as
phosphoprotein detection. chemiluminescence, are exquisitely sensitive, while
others, such as those using colorimetric substrates have
lower sensitivity. 45
Antibody Applications

The appropriate working concentration of the primary

antibody depends on the binding characteristics of the Nice to know
primary antibody and is also greatly affected by the type
When extra bands appear below that of the
of detection system that is employed.
desired protein, the most likely explanation
is that they originated from proteolytic
If the proper primary antibody dilution for a colorimetric breakdown of the desired protein. This can
detection system is substituted into a chemiluminescent be prevented by treating the sample with
detection system without further optimization, it is protease inhibitors during tissue or cell sample
very common to see a high background signal. It is preparation. Also, if the sample buffer does not
necessary to perform an additional dilution series with contain sufficient SDS and/or reducing agent
the primary antibody to determine the optimal dilution (DTT, 2-mercaptoethanol, BMS), the protein
for this more sensitive detection system. Likewise, the may not be fully dissociated into its subunits,
proper primary antibody dilution for a chemiluminescent reduced or denatured. Hence, extra bands may
appear above the desired protein on the gel.
detection system may give an undetectably low signal for
Heating the sample in sample buffer at 65°C
colorimetric detection.
for 10-15 min immediately prior to loading
can reduce these non-covalent interactions
Other labels include: or disulfide linkages. Use of an irreversible
1. 125I-labeled secondary antibody, which can be detected reducing agent such as TCEP, instead of DTT or
using a photographic film. mercaptoethanol, may also be helpful.
2. 125I-labeled Protein A. In this case Protein A is used
instead of a secondary antibody, as it will bind to the Sometimes, the bands are due to specific
Fc region of IgG molecules. recognition of other epitopes in the sample by
3. Gold-labeled secondary antibody. The minute gold the primary antibody. For example, an antibody
particles are directly visible as a red color when they made against a peptide coupled to BSA
are bound with the secondary antibody to the primary may recognize traces of BSA in the sample.
Homologous proteins may also be recognized
antibody.
by the antibody. When using a polyclonal
4. Biotinylated secondary antibody. In this case the
serum, some bands may be due to the presence
blot is incubated with the secondary antibody, and
of antibodies produced as a result of animal’s
then incubated with enzyme-conjugated avidin that exposure to similar antigens in its life. It is best
binds strongly to the biotin. This system will give an to run a normal serum control to determine the
enhanced signal, as multiple biotin molecules can be specificity of any antibody.
attached to a single antibody molecule. The enzyme
used is usually alkaline phosphatase or horseradish
peroxidase.

Refer to manufacturer instructions for specific protocols

for detection with different substrates.
Western blotting

46
 Antibody Applications
3.4.8 Common errors
Here are some examples of commonly seen artifacts in
Western blotting:

Example 1: Example 2:
kDa kDa Background is too
260 260 high; this blot
160 160 was not properly
110 washed.
110
80 80
60 60
50 50
40 40
30 30
20 20

Gel run is “smiling.” This is caused by too high

a voltage. Also, target bands are bleached,
caused by using the detection reagent at too
high of a concentration. Background is too
high, caused by using the primary and/or
secondary antibody at too high a concentration.

Example 3:
kDa
260 Smears are shown kDa
160 instead of distinct 260
bands, caused by 160
110 loading too much
80 sample into each 110
well. 80
60
50 60
40 50
40
30
20 30
20

Bleached bands are shown. Chemiluminescent

substrate was used at too high a concentration,
or incubated too long before exposure. (Target is
at 180 kDa, bleached bands are at ~60 kDa.)

Western blotting

47
Antibody Applications

3.4.9 Troubleshooting:
Problem Possible Cause Solution
Streaking of blots Excess protein loaded onto gel. Accurately measure protein concentration, and load less protein.
Smudgy or fuzzy Gel improperly equilibrated and Check gel equilibration times.
blots shrinking during blotting
Poor staining of blot Ineffective transfer Carefully remove air bubbles when making sandwich.
with Ponceau S
Check that excess temperatures are not reached during electroblotting producing
bubbles, or gel/membrane distortion.
No staining of blot Proteins did not transfer during Check equipment (boxes, tops, power source).
with Ponceau S Western blotting
Check that the gel and membrane Check that the membrane is on the right side of the gel.
make proper contact during blotting
No signal Protein degraded during storage of Use fresh blots.
blots prior to detection
Wrong secondary antibody Check for appropriate secondary antibody.
Exposure time too short Increase length of exposure.
Insufficient antigen Load more antigen on gel, or concentrate sample prior to loading, or use
immunoprecipitation to increase amount of target protein run in gel.
Antigen may have been destroyed Check that antigenicity is not destroyed by treatment for electrophoresis.
Detection system Increase (and optimize) concentration, incubation times, and temperatures of
primary antibody.
Increase (and optimize) reagent concentration and incubation times, for your
specific application.
Check that the detection reagents are being stored correctly and used as
recommended.
Low affinity primary antibody Remove Tween® from antibody buffer.
Chemiluminescent substrate has Prepare a small amount of working solution (1 mL), go into a dark room and
lost activity add 1 mL of HRP conjugate. Visible blue light should be observed.
Membrane has been stripped and There may be antigen loss during reprobing.
reprobed.
Weak signal See under “No signal”.
Insufficient protein on the gel Load more protein on gel, or concentrate sample prior to loading, or use
immunoprecipitation to increase amount of target protein run in gel.
Expose film for an extended period (1-2 hours).
Poor protein transfer onto the membrane--optimize transfer conditions, such as
transfer buffer pH, membrane type and voltage.
Nonspecific bands Antibody (primary or secondary) Reduce antibody concentrations.
concentrations are too high
SDS can cause nonspecific binding to Wash blots well after transfer, using agitation.
immobilized protein bands
Do not use SDS during procedure.
Diffuse bands Antibody (primary or secondary) Reduce antibody concentrations.
concentrations are too high
Too much protein is loaded on the gel Reduce the amount of protein loaded.
Black blots with Antibody (primary or secondary) Reduce antibody concentrations, especially the HRP conjugate. Signals that
white bands or signal concentrations are too high decrease quickly and white bands are an indication that the HRP is “burning out”.
that decreases quickly
Western blotting

48
 Antibody Applications
Problem Possible Cause Solution
Partialy developed Incomplete transfer of proteins from Eliminate air bubbles between the gel and membrane during transfer.
area or blank areas the gel
Incubate membranes separately to ensure that membrane strips are not covering
one another during incubations.
High background Antibody (1° Ab or 2° Ab) Decrease antibody dilutions of either primary or secondary antibody
uniformly distributed concentrations are too high or use shorter incubation times.
Wrong blocking buffer was used Compare with other blocking buffers.
Insufficient blocking of nonspecific Increase the concentration of protein in the blocking buffer.
sites
Optimize blocking time and/or temperature Add Tween®-20 to blocking buffer.
A concentration of 0.05% Tween® 20 is recommended.
Cross reactivity of antibody with other Use a different blocking buffer. Dilute primary antibody in buffer with no other
proteins in blocking buffer proteins.
Do not use milk to block membranes when using a avidin-biotin system.
Use a freshly prepared solution of blocking agent.
Insufficient washing Increase number of washes and the volume of buffer used.
Add Tween® 20 to to wash buffer if it’s not already included.
Increase concentration of Tween® 20 blocking solution to 0.1% Tween® 20.
High background Exposure time is too long Reduce the time the blot is exposed to film.
uniformly distributed
Expose the film for a minimum period (an initial 15 seconds exposure may be all
that is required). If exposure time is too short to be convenient, reduce antibody
concentrations.
Leave blots in the cassette for 5-10 minutes before re-exposing to film.
Detection reagents causing high Rewash blots twice for 10 minutes in wash buffer and repeat detection steps.
background
Excess detection reagents on blots. Drain well by absorbing the excess on blotting
paper before placing blots in film cassettes.
Membrane problems Make sure membranes are wetted thoroughly and kept wet throughout the
procedure.
Use agitation during all incubations. Handle membranes carefully - damage to the
membrane can cause nonspecific binding.
Do not handle membrane with bare hands. Use gloves!
Always wear clean gloves or use forceps.
Contamination or growth in buffers Prepare fresh buffers.
Contaminated blocking equipment Clean or replace all equipment.
Blotchy or speckled Antibody (primary or secondary) Optimize antibody concentrations. The primary/secondary antibody can cause high
backgrounds concentrations too high background if the concentrations
used are too high.
Aggregate formation in the HRP Filter the conjugate through a 0.2 µL filter.
conjugate can cause speckling
Wrong blocking buffer was used Compare with other blocking buffers.
Insufficient blocking of nonspecific Increase the concentration of protein in the blocking buffer.
sites
Optimize blocking time and/or temperature.
Add Tween® 20 to blocking buffer. A concentration of 0.05% Tween® 20 is
recommended.
Western blotting

Reference:
Protein Blotting Handbook: Tips and Tricks. EMD Millipore. 2012. Billerica, MA. (Literature No. TP001EN00).
49
Antibody Applications

3.4.10 A Note on Membrane Selection

The type of membrane used for blotting can influence the following:
• Protein binding capacity
• Requirement for prewetting with alcohol
• Ability to perform multiple stripping and reprobing experiments
• Protein visualization
• Long-term blot storage
• Signal-to-noise ratio
Polyvinylidene fluoride (PVDF) and nitrocellulose are the two membrane types most commonly used in Western blotting
applications

Comparison of PVDF and nitrocellulose membrane attributes and applications

Attributes/Applications Nitrocellulose PVDF
Physical strength Poor Good
Protein binding capacity 80 – 100 μg/cm2 100 – 300 μg/cm2
Solvent resistance No Yes
Western transfer Yes Yes
Total protein stain Colloidal gold Colloidal gold
Ponceau-S red Ponceau-S red
Amido black Amido black
India ink India ink
Sypro® blot stains Coomassie® Blue dye
Detection Chromogenic Chromogenic
Chemiluminescent Chemiluminescent
Fluorescent Fluorescent
Radioactive Chemifluorescent Radioactive
Double-blotting method No Yes
Rapid immunodetection No Yes
Western reprobing Yes Yes
Edman sequencing No Yes
Amino acid analysis Yes Yes
Binding in the presence of SDS Poor Good
On-membrane digestion or mass spectrometry No Yes
Direct MALDI-TOF MS analysis No Yes
Data can be archived No Yes

Technology Highlight

SNAP i.d.® 2.0 System

Developed to meet the needs of our Western blotting customers, the SNAP i.d.®
2.0 system produces blots of a very high quality every time – in record time!
Unique vacuum-driven technology and a built-in flow distributor actively drive
reagents through the membrane, ensuring even distribution. Two blot holder sizes
accommodates mini (7.5 x 8.4 cm) or midi (8.5 x 13.5 cm) (W x L) size gels and two
blot holders can be run in parallel. Thus, you can quickly optimize conditions and
Description Cat. No.
greatly increase your protein detection throughput. SNAP i.d.® 2.0 Protein Detection SNAP2MINI
System-Mini (7.5 x 8.4 cm)
Typically, researchers lack the time to optimize their blotting protocols. By SNAP i.d.® 2.0 Protein Detection SNAP2MIDI
System-Midi (8.5 x 13.5 cm)
Western blotting

shortening the time required for blocking, washing and antibody incubations to SNAP i.d.® 2.0 Protein SNAP2MM
30 minutes, the SNAP i.d.® 2.0 system allows you to optimize your Detection System-Mini and Midi
(7.5 x 8.4 cm and 8.5 x 13.5 cm)
immunodetection conditions for the highest quality results.

50
 Antibody Applications
3.5 Enzyme-linked Immunosorbent 3.5.2 Sandwich ELISA
Assays (ELISA) The two-antibody sandwich ELISA is used to determine
the antigen concentration in unknown samples, and is
3.5.1 Introduction one of the most useful immunoassay techniques. The
3.5.2 Sandwich ELISA sandwich ELISA is fast and accurate, and, if a purified
3.5.3 Competitive ELISA
antigen standard is available, the assay can be used to
3.5.4 Substrates
generate a dose-response curve and to determine the
3.5.5 Quantification
3.5.6 Troubleshooting absolute amount of antigen in an unknown sample.

To use this assay, a “sandwich” is created as follows:

• A purified, target-specific antibody (the “capture”
3.5.1 Introduction antibody) is bound to a solid phase - typically
Enzyme-Linked Immunosorbent Assay (ELISA) is a
attached to the bottom of a plate well.
technique that combines the specificity of antibodies
• Sample (which may contain an unknown amount of
with the sensitivity of simple enzyme assays. By using
antigen) is added and allowed to complex with the
antibodies or antigens coupled to an easily assayed
bound antibody. Unbound products are removed by
enzyme that possesses a high turnover number, ELISAs
washing.
can provide a useful measurement of antigen or antibody
• A labeled second antibody (the “detection” antibody) is
concentration. There are two commonly used ELISA
added and allowed to bind to the antigen.
formats: Sandwich ELISA and Competitive ELISA. These
are illustrated and described below.

Sandwich ELISA
Figure 10.
Differences between a
sandwich ELISA and a E E
competitive ELISA.

Add Add Add Add

Antigen 2nd 1° Ab (detection) Enzyme-Labeled 2° Ab Substrate

-+
Standard Curve

Measure Change
OD

in Absorbance Signal
ng of antigen

Dilutions

Competitive ELISA

Coat Well with

1st 1° Ab (capture)
E E E E
E E

Add Add Add

Standards & Samples Enzyme-Labeled Antigen Substrate

+-
Standard Curve

Measure Change
OD

ELISA

in Absorbance Signal
ng of antigen
Dilutions
51
Antibody Applications

The assay is quantitated by measuring the amount of 3.5.3 Competitive ELISA Assays
labeled second antibody bound to the matrix, through When two “matched pair” antibodies are not available
the use of a colorimetric substrate. for your target, another option is the competitive ELISA.
As the name suggests, this assay makes use of the
Major advantages of this technique are that the antigen competition between reagents in order to determine
need not be purified prior to use, and that these assays the relative amount of an antigen. In this assay, one
are very specific. reagent must be conjugated to a detection enzyme, such
as horseradish peroxidase. The enzyme may be linked to
However, there is a disadvantage, is that not all either the immunogen or the primary antibody. Although
antibodies can be used in this application. Monoclonal there are several different configurations for competitive
antibody combinations must be qualified as “matched ELISAs, below is an example of one such configuration.
pairs” - meaning that they can recognize separate
epitopes on the antigen so they do not hinder each A competitive ELISA can be performed as follows:
other’s binding. • An unlabeled purified primary antibody is used to coat
the wells of a 96-well microtiter plate.
The amount of the capture antibody bound to the solid • This primary antibody is incubated with unlabeled
phase can be adjusted easily by dilution or concentration standards and unknowns, and the reaction is allowed
of the antibody solution. The avidity of the antibodies to reach equilibrium.
for the antigen can only be altered by substitution with • A conjugated immunogen is added. This conjugate will
other antibodies. The specific activity of the second bind to the primary antibody wherever its binding sites
antibody is determined by the number and type of are not already occupied by unlabeled immunogen.
labeled moieties it contains. Thus, the more immunogen in the sample or standard,
the lower the amount of conjugated immunogen
The sensitivity of the sandwich ELISA is dependent bound.
on four factors:
1. The number of molecules of the first antibody that are The competition is allowed to reach equilibrium and the
bound to the solid phase. plate is “developed” with a substrate (discussed below).
2. The avidity of the first antibody for the antigen. The chemiluminescence or color change is measured to
3. The avidity of the second antibody for the antigen. indicate the amount of antigen present in the sample.
4. The specific activity of the second antibody.
An advantage to the competitive ELISA is that unpurified
primary antibodies may be used. As mentioned
Nice to know previously, there are multiple configurations, with a
The sandwich ELISA requires two antibodies competing labeled immunogen being common. For other
that bind to epitopes that do not overlap configurations of competitive ELISAs, see Antibodies. A
on the antigen. This can be accomplished Laboratory Manual, by Ed Harlow and David Lane (Cold
with either two monoclonal antibodies that Spring Harbor Laboratory, Cold Spring, New York, 1988).
recognize discrete sites or one batch of
affinity-purified polyclonal antibodies.
ELISA

52
 Antibody Applications
3.5.4 Substrates for ELISA acid) (ABTS), O-phenylenediamine (OPD), and 3,3’,5,5’-
Unlike Western blots, which use precipitating substrates, tetramethylbenzidine base (TMB), which yield green,
ELISA procedures use substrates that produce soluble orange, and blue colors, respectively.
products. Popular enzymes are alkaline phosphatase (AP)
and horseradish peroxidase (HRP). Ideally the enzyme
substrates should be stable, safe, and inexpensive.
Watch Out
These enzymes can convert a colorless substrate to a Sodium azide is an inhibitor or horseradish
colored product, e.g., p-nitrophenylphosphate (pNPP), peroxidase. Do not include sodium azide in
which is converted to the yellow p-nitrophenol by buffers or wash solutions if an HRP-labeled
alkaline phosphatase. Substrates used with peroxidase antibody will be used for detection.
include 2,2’-azo-bis(3-ethylbenzthiazoline-6-sulfonic

Chromogenic ELISA substrates

Alkaline Phosphatase
Buffer/ Second Reagent to Wavelength for
Substrate Substrate stop reaction Color of Product quantitation
p-Nitrophenyl Phosphate (pNPP) Na2CO3, pH 9.8 with NaOH, 2M Yellow 405 nm
MgCl2

Horseradish Peroxidase
Buffer/ Second Reagent to Wavelength for
Substrate Substrate stop reaction Color of Product quantitation
3,3’,5,5’-Tetramethyl-benzidine (TMB) 30% Hydrogen 1 M Sulfuric Acid Blue 450 nm
Peroxide (H2O2) (H2SO4)
o-Phenylene Diamine (OPD) Citrate Phosphate Sulfuric Acid (H2SO4) Orange-Brown 492 nm
Buffer, 0.02% H2O2
2,2’-azinodiethyl-benzthiazoline Citrate Phosphate 20% SDS / 50%DMF Green 410 nm, 650 nm
sulfonate (ABTS) Buffer, 30% H2O2

The most sensitive ELISA detection method involves the enzyme catalyzing the reaction of a chemiluminescent
substrate to generate light. For example, Luminata™ ELISA HRP substrates, when catalyzed by HRP, generate a signal
detected using a luminometer.

Chemiluminescent ELISA substrates

Horseradish Peroxidase
Luminata™ Forte ELISA HRP Substrate (Cat. No. ELLUR0100)
Luminata™ Crescendo ELISA HRP Substrate (Cat. No. ELLUF0100)
ELISA

53
Antibody Applications

3.5.5 Quantification points at the top or bottom of the range tested may be
A plate reader or luminometer set to measure at the dropped to get a good fit.
appropriate wavelength is used to quantify the signal. By
comparing the specific signal in a sample to the standard An example showing ELISA results is illustrated
curve, the relative amount of the antigen in the sample below. A standard curve was generated using known
can be estimated (see example below). concentrations of the antigen and measuring absorbance
at 405 nm (blue line). A sample yielded an average
To create a standard curve, known concentrations absorbance of 1.42 nm. Extrapolating to the standard
of antigen are plotted on the X-axis and the curve, the sample contains ~10 ng/mL of the antigen
corresponding absorbance on the Y-axis. The standard (dashed red line).
curve should result in a graph that shows a direct
relationship between antigen concentrations and the 2.25
2.00
corresponding absorbances. In other words, the greater 1.75
the concentration of the antigen in the sample, the 1.50 Sample 1.42 nm

A405 (nm)
higher the absorbance. The concentration of the antigen 1,25
1.00
in unknown samples may be determined by plotting 0.75
the sample absorbance on the Y-axis, then drawing a 0.50
0.25
horizontal line to intersect with the standard curve.
0.00
A vertical line dropped from this point intersects the 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5
X-axis at the concentration of antigen in the unknown Antigen Standard [ng/mL]

sample. An alternative approach is to enter the data

into a computer program curve fitting software, such Figure 11.
as MILLIPLEX® Analyst 5.1 software. A good fit can be ELISA standard curve and sample analysis.
obtained with a linear regression analysis. Some data

3.5.6 Troubleshooting:
Problem Possible Cause Solution
No signal Wrong test reagents used Ensure that only the reagents for the specific test-lot are used.
Test reagents damaged Don’t use the test kit after expiration date.
Weak signal Test reagents used in a wrong Control used test dilutions carefully (usually a dilution factor of
dilution 100 is used).
Wrong filter (wavelength) Check your wavelength in your microtiter plate photometer.
Incubation time too short Check the information of incubation times of the lot in the
temperature too low product data sheet. (The incubation time of the enzyme substrate
is applied for temperatures from 20 to 28°C); extend the
substrate incubation time, if absorption is below 1.0
Reagents not at right temperature Make sure that the reagents used for day 2 have reached room
temperature (20 to 28°C) before using within the test kit.
Sodium azide, mercaptoethanol or Only use samples which contain no or low concentrations
DTT can interfere with peroxidase (< 0.1 %) of sodium azide, mercaptoethanol or DTT.
activity at high concentrations
High signal Test reagents used in a wrong Check used test dilutions carefully (usually a dilution factor of
dilution 100 is used).
Incubation time too long Check the information of incubation times of the lot in the
temperature too high product data sheet. (The incubation time of the enzyme substrate
is applied for temperatures from 20 to 28°C); shorten the
substrate incubation time, if absorption is above 3.0
ELISA

54
 Antibody Applications
Problem Possible Cause Solution
High background Insufficient washing steps Wash plate carefully and remove the liquid after each washing
(blank) carefully.
Contamination of the washing Confirm that the water is not contaminated. Use always double
solution distilled water for the reconstitution and dilution of the washing
solution.
Contamination of reagents or vials/ Avoid pipetting directly out of the reagent vials, if test reagents
tubes from previous experiments should be used in further measurements. (Oxidative active
contaminants can influence the enzyme substrate by non-
specific color development).
Test reagents (antibody- and Check used test dilutions for antibody and enzyme conjugate
enzyme conjugate) used in wrong carefully (usually a dilution factor of 100 is used).
dilutions
Low precision Non-homogeneous samples e.g. Check that the samples are taken, prepared and stored according
(=random error) cloudy solution, particles in the to a recommended sample procedure (polypropylene tubes,
sample storage of clear samples at -20°C).
Insufficient mixing of samples and Mix samples and standards before pipetting carefully.
standards
Variation in pipetting Check your pipettes and calibrate if necessary.
Carry over between samples and/ Change pipet tips after each pipetting.
or standards
Insufficient mixing of reagents Mix reagents on the test plate after pipetting by moving the
during incubation test plate carefully; use an orbital microtiter plate shaker on the
recommended test steps for optimal mixing of reagents.
Insufficient washing Check that the automatic microtiter plate washer is working
correctly; residues of liquids must be removed completely after
each washing step.
Evaporation of liquids Check the contact of the cover seal with the plate during the
incubation steps.
Calculated data are Calculation of the dilution factor is Check the dilution factor used for the sample dilution within the
too high or too low not correct data calculation.
(=systematic error,
Modification of the test procedure Follow the instructions in the product data sheet carefully
deviation of data
(incubation time, dilution etc.).
from “typical data”)
Incorrect sample treatment Check that the samples are taken, prepared and stored according
to a recommended sample procedure (polypropylene tubes,
storage of clear samples at -20°C).

Technology Highlight

Bring your biomarkers to life.

The best, most relevant ELISAs and RIAs
for your protein research.
A complete picture of metabolic syndrome, inflammation or neurological
disorders is more than the sum of individual analytes. To help you put the
pieces together, we’ve built the largest portfolio of assays for soluble and
intracellular biomarkers. Our manufacturing of ELISAs and RIAs is the gold
standard, giving you the same accuracy and precision in every lot, backed
by the same, unwavering technical support.

Cytokine / Chemokine ELISA Kits

Neuroscience: Neuropeptide & Neurodegeneration ELISA Kits
Metabolic/Endocrine ELISAs Cell Signaling ELISAs
Transcription Factor Assay (TFA) ELISAs
Radioimmunoassays (RIAs)
ELISA

55
Antibody Applications

3.6 Multiplexed Bead-based Bead Set 21 Bead Set 26

Detection 6.5 Microns

3.6.1 Introduction
3.6.2 Technology
3.6.3 Troubleshooting
The bead is

10 Con
Dy
impregnated

Un cen
e
with the

iqu tra
dye mixture

e In tio
3.6.1 Introduction

fra ns
red
Multiplex analysis, as the name suggests, is the
10 Unique Red Dye Concentrations
technique of assaying multiple analytes in a single assay.
Multiplexed detection is a technology for biomarker
screening and protein analysis that enables researchers Figure 12.
xMAP® technology: quantitate up to 100
to simultaneously investigate the expression of multiple different analytes per sample/well.
inter- or intracellular proteins, total or phosphorylated,
involved in cellular, tissue, or organismal function.

The rapidly growing knowledge base in drug discovery

Microspheres are dyed with varying concentrations of
and protein research has placed increased pressure
two red fluorescent dyes to create up to 100 distinct
on researchers to rapidly obtain and analyze systems-
colors. Thus each microsphere in an assay has a ‘spectral
level data sets connecting proteins and pathways to
address’ based on the dye concentrations that can then
biological states. Increasingly, this information is difficult,
be read and identified by the Luminex analyzer. These
impractical, or cost-prohibitive to obtain using traditional
microspheres are coated with a capture antibody to
“singleplex” protein detection methods, such as ELISA or
specifically bind a target ‘analyte’ in the sample. A second
Western blotting. Since multiplexing allows detection &
reporter-tagged antibody is added to complete the
measurement of many analytes in a single complicated
‘sandwich’ ELISA for readout (see Figure 13).
and heterogeneous sample, the technique proves
particularly useful when analyzing serum, cerebrospinal
APD APD 633 nm
fluid, glandular secretions, or other physiological
samples.

3.6.2 Technology
Techniques for multiplexed protein detection most Luminex Beads
often involve presentation of antibodies to antigens
in a sandwich immunoassay fashion and are either
Capture
immobilized on chips as “planar arrays” or conjugated Figure 13.
Antibody
to micro beads as “suspension arrays.” The extremely Principle of Luminex
xMAP® bead-based
versatile and sensitive free-floating bead arrays are based immunodetection.
on the xMAP® system developed by Luminex Corporation. Analyte

This system is the combination of three core Biotinylated

technologies: xMAP® microspheres, Luminex analyzers, Detection
Antibody
and analysis software. Streptavidin-
Phycoerythrin

PMT

532 nm
Multiplex

56
 Antibody Applications
Two types of Luminex analyzers can be used to perform multiplex bead-based assays. Luminex 200™ and FLEXMAP
3D® systems are flow cytometry-based instruments that integrate lasers, optics, advanced fluidics and high-speed
digital signal processors (Figure 14A). The MAGPIX® instrument is a CCD-based system that integrates xMAP® capture
and detection components with the speed and efficiency enabled by magnetic beads (Figure 14B).

A. Luminex 200™ & FLEXMAP 3D® B. MAGPIX® LED/Image-based Analysis

Flow Cytometry-based Analysis

Monolayer
Monolayer beadsbeads
Sheath
Sheath fluid fluid
hydrodynamic
hydrodynamic
focusing
focusing of of
sample
sample

Magnetic
Magnetic Capture
Capture
Interrogate
Interrogate bead bead Interrogate
Interrogate label label 0.5 sec
0.5 sec
with with red laser
red laser with with
greengreen
laser laser dwelldwell
time time
(635 nm) nm)
(635 (525 (525
nm) nm)

10 msec
10 msec
dwelldwell
time time

Identify
Identify bead bead Quantify
Quantify Interrogate
Interrogate bead bead Interrogate
Interrogate label label
region basedbased
region on on binding
binding events
events with red
with red LED LED Identify
Identify and and with green LED LED
with green
internal
internal dye dye (635 (635
nm) nm) quantify with with (525 (525
quantify nm) nm)
concentrations
concentrations CCD imager
CCD imager

Figure 14.
Two types of Luminex instruments, either flow cytometry-based (A) or CCD imaging-based (B), are available for acquiring
data from multiplexed bead-based immunoassays using xMAP® technology.

The readout from the instruments includes a “bead map” to identify analytes present and standard curves for panel
analytes to determine concentration. Since both classification and reporter readings are made on each individual
bead simultaneously, this allows for precise multiplex assay results within the same small sample; which is a vast
improvement over traditional ELISAs.

Analyzing data from multiplexed biomarker assays can be difficult when working with diverse sample and analyte
types. This diversity can lead to a wide range of possible analyte levels and assay signal intensity with respect to those
analyte levels, both of which are not always easy to predict or determine accurately. MILLIPLEX® Analyst 5.1 software
was designed to generate the most meaningful quantitative analyte data with a focus on data derived from the low
and high ends of standard curves. Data in these regions can be important and are commonly missed by existing
multiplex data analysis packages.
Multiplex

57
Antibody Applications

In developing the new curve fitting algorithms for Human Cytokine/Chemokine 39-Plex Magnetic Bead
MILLIPLEX® Analyst 5.1 software, simulations were run Standard Curves in Matrix
100,000
on over 600 data sets using actual experimental standard
curves in order to determine the curve fits that would 10,000

Median Fluorescence
give the lowest CVs at the low end and high ends of the

Intensity (MFI)
1,000
curves and that would work well even with standard
100
curves of low quality.
10

Figure 15. 1
Analytically validated fixed

10

00

00
10

,00
1,0

0,0
standard curves in

10

10
MILLIPLEX® map Concentration (pg/mL)
multianalyte panels
(based on Luminex® EGF Eotaxin FGF-2 FIt-3L IL-13 IL-17
xMAP technology) enable Fractakine G-CSF GM-CSF GRO MCP-1 MDC
reproducible quantitation. IFNα2 IFNγ IL-1α IL-1β MIP-1α sIL-2Rα
IL-1ra IL-2 IL-3 IL-4 TNFα VEGF
IL-5 IL-6 IL-7 IL-8 IL-15 IP-10
IL-9 IL-10 IL-12(p40) IL-12(p70) MCP-3 MIP-1β
sCD40L TNFβ TGFα

3.6.3 Troubleshooting - Multiplexed Bead-based Assays:

Problem Possible Cause Solution
Insufficient Plate washer aspiration Adjust aspiration height according to manufacturers’
Bead Count height set too low instructions.
Bead mix prepared inappropriately Sonicate bead vials and vortex just prior to adding to bead mix
bottle according to protocol. Agitate bead mix intermittently in
reservoir while pipetting this into the plate.
Samples cause interference due to See above. Also sample probe may need to be cleaned with
particulate matter or viscosity Alcohol flush, Back flush and washes; or if needed probe should
be removed and sonicated.
Probe height not adjusted correctly Consult manufacturer instructions. When reading the assay on
a Luminex 200™ instrument, adjust probe height according to
the protocols recommended by Luminex to the kit solid plate
using 4 alignment discs. When reading the assay on a FLEXMAP
3D™ instrument, adjust probe height according to the protocols
recommended by Luminex to the kit solid plate using one
alignment disc. When reading the assay on a MAGPIX® system,
adjust probe height according to the protocols recommended by
Luminex to the kit solid plate using two alignment discs.
Background is Background wells were contaminated Avoid cross-well contamination by using sealer appropriately and
too high pipeting with multichannel pipets without touching reagent in
plate.
Insufficient washes Increase number of washes.
Beads not in Luminex instrument not calibrated Calibrate Luminex instrument based on Instrument
region or gate correctly or recently Manufacturer’s instructions, at least once a week or if
temperature has changed by >3°C.
Gate Settings not adjusted correctly Some Luminex instruments require different gate settings than
those described in the kit protocol. Use Instrument default
settings.
Wrong bead regions in protocol template Check kit protocol for correct bead regions or analyte selection.
Incorrect sample type used Samples containing organic solvents or if highly viscous should
be diluted or dialyzed as required.
Instrument not washed or primed Prime the Luminex instrument 4 times to remove air
bubbles,wash 4 times with sheath fluid or water if there is any
residual alcohol or sanitizing liquid.
Beads were exposed to light Keep plate and bead mix covered with dark lid or aluminum foil
during all incubation steps.
Multiplex

58
 Antibody Applications
Problem Possible Cause Solution
Signal for Incorrect or no detection Add appropriate Detection Antibody and continue.
whole plate
Antibody was added
is same as
background Streptavidin-Phycoerythrin was not Add Streptavidin-Phycoerythrin according to protocol.
added
Low signal for Incubations done at inappropriate Assay conditions need to be checked.
positive Lysate temperatures, timings or agitation
Control
Sample signals Calibration target value set too high With some Luminex Instruments, default target setting for RP1
too high and calibrator is set at High PMT. Use low target value for calibration
saturated and reanalyze plate.
Plate incubation was too long with Use shorter incubation time.
Lysate Control and samples
Samples contain analyte concentrations Samples may require dilution and re-analysis for just that
higher than the assay dynamic range particular analyte.
Sample signals Samples contain no or below detectable If below detectable levels, it may be possible to use higher sample
too low levels of analyte volume. Check with technical support for appropriate protocol
modifications.
High Variation Multichannel pipet may not be calibrated Calibrate pipets.
in samples and/
Plate washing was not uniform Confirm all reagents are removed completely in all wash steps.
or standards
Samples may have high particulate See above.
matter or other interfering substances
Plate agitation was insufficient Plate should be agitated during all incubation steps using a
vertical plate shaker at a speed where beads are in constant
motion without causing splashing.
Cross-well contamination Check when reusing plate sealer that no reagent has touched
sealer.
Care should be taken when using same pipet tips that are used
for reagent additions and that pipet tip does not touch reagent
in plate.

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59
Antibody Applications

3.7 Immunohistochemistry/ The main steps in IHC/ICC are as follows:

• Specimen Preparation
Immunocytochemistry
• Antigen Retrieval
3.7.1 Introduction • Antibody Staining
3.7.2 Specimen Preparation • Antibody Detection

Key Steps
3.7.3 Antigen Retrieval
3.7.4 Antibody Staining 3.7.2 Specimen Preparation
3.7.5 Antibody Detection
The ability to successfully visualize and interpret IHC
3.7.6 Counterstaining
staining is dependent upon the quality of the histological
3.7.7 Troubleshooting
sections used. There are several key preparatory steps
3.7.1 Introduction that must be done prior to antibody incubation and
Immunohistochemistry (IHC) refers to the process tissue staining:
of detecting antigens (e.g., proteins) in cells of a • Fixation
tissue section by exploiting specific antibody-antigen • Embedding
interactions. IHC takes its name from the root words • Sectioning
“immuno,” in reference to antibodies used in the
procedure, and “histo,” meaning tissue. In comparison, These steps, and the antigen retrieval step that follows,
immunocytochemistry (ICC) differs in the root word must be done with proper care in order to obtain useful
“cyto”, meaning cell, and is performed on cultured or results. Variations in technique can greatly impact
isolated cells instead of tissue. IHC and ICC are widely staining results. These steps are discussed in some detail
used in basic research to understand the distribution and below:
localization of biomarkers and differentially expressed
proteins in biological tissues and cells, respectively. Fixation
Fixation is a chemical or physical process that protects
and preserves (i.e. “fixes”) biological tissues in a state
A. B.
that is closest to their natural state at any given time
in order to permit further processing. Reagents used
for fixation, or “fixatives,” achieve this in three ways:
they stop proteolysis, enhance structural stability, and
inhibit putrefaction by microorganisms. Major classes
C. D. of chemical fixatives include: aldehydes, alcohols,
mercurials, oxidizing agents, and picrates. Physical
fixation commonly refers to freezing tissue, which is
generally followed by chemical fixation at some point
prior to subsequent processing.

Figure 16.
IHC validation of anti-desmoglein antibody (1:1,000,
Cat. No. MABT118) using human tonsil tissue. Human
tonsil tissue was analyzed in IHC following antigen retrieval
(HRP-DAB detection). As expected, membrane/cell junction
immunoreactivity was observed in the stratified squamous
epithelium (A) as well as the epithelial cells lining the tonsillar
crypts of the human tonsil (C). Treatment of the same tissues
with negative control reagent (no primary antibody) resulted
in no detectable HRP-DAB signal (B, D). Counterstaining of the
tissues with hematoxylin stains the cell nuclei blue.
IHC / ICC

60
 Antibody Applications
To achieve this, tissue blocks, tissue sections, cells in then the paraffin must be removed from the tissue
culture, or smears are either immersed in a fixative fluid, to allow the water-based buffers and antibodies to
or in cases where whole animal systems are studied, the penetrate.
animal is perfused with fixative via its circulatory system.
In the case of cells in culture, cell preparations are either Sectioning
submerged or simply air-dried. Depending on the embedding medium used (i.e. paraffin
or aqueous freezing media) subsequent sectioning
Fixatives stabilize cells and tissues, thereby protecting may be performed on a microtome or cryostat. A
them from the rigors of subsequent processing and microtome is generally used at room temperature on
staining techniques. Fixatives may work by several paraffin-embedded tissue blocks to produce ultrathin
means: formation of crosslinks (e.g., via aldehydes such sections, ranging from 1-60 µm. A cryostat is basically
as glutaraldehyde or formalin), protein denaturation a microtome in a climate-controlled cabinet, which
by coagulation (e.g., via acetone and methanol), or a keeps all of the sectioning equipment and tissue blocks
combination of these. Fixation strengths and times between -10 and -30°C. Optimal section thickness is
must be optimized so that antigens and cellular around 5 µm (if achievable), which typically provides the
structures can be retained and epitope masking is least amount of cell overlap.
minimal. Requirements for fixation can vary widely
between tissues. For immunological studies, fixation
is especially imperative to ensure the adequacy of the Nice to know
specimen and target antigens. Tissues have differing The less a tissue is processed (i.e., shorter
protein content and structural arrangement; thus they fixation, looser embedding, less processing time),
vary in their ability to retain their structure without the less there is need for antigen retrieval.
significant fixation. Incorrect specimen preparation
can block or impede antigen labeling in tissue and
3.7.3 Antigen Retrieval
cells. Unfortunately, the methods that are best for
To facilitate the immunological reaction of antibodies
the preservation of tissue structure do so by altering
with antigens in fixed tissue, it may be necessary to
proteins, thereby masking some epitopes and sometimes
unmask or “retrieve” the antigens through pretreatment
preventing the detection of the desired target protein.
of the specimens. There are many forms of antigen
In cases of failure, it is important to experiment with
retrieval (sometimes called antigen recovery), and
different fixatives and antigen retrieval methods prior to
different antigens and different antibodies will require
“giving up” on a specific stain.
different antigen retrieval methods. Antigen retrieval
has been shown to increase reactivity of the majority
Embedding
of antigens in tissues. The use of antigen retrieval
Most samples used in immunostaining are embedded in
in immunocytochemistry is less common, however
paraffin because it provides for excellent morphological
depending upon the particular antibody/antigen
detail and resolution. Modern “paraffin” is typically
combination it can be performed on cell preparations,
a mixture of paraffin wax and resin. It is an excellent
although the length of time and intensity is typically
embedding medium because it can be heated to liquid
much less than for tissue. Antigen retrieval includes
state, dissolved by xylene for infiltrating the tissue and
a variety of methods by which the availability of the
then relatively quickly turned to a solid state again for
antigen for interaction with a specific antibody is
maximum structural support during sectioning. Typically,
maximized. The most common techniques are:
small blocks (10 x 10 x 3 mm) of tissue are fixed for up
• Enzymatic Digestion
to 24 hrs. The most common fixatives used in paraffin
• Heat Induced Epitope Retrieval (HIER)
sections are formalin-based. These fixatives are well
• Citric Acid Incubation
tolerated by the tissues and achieve good penetration.
(See Appendix for Recipes of Common Fixatives).
Enzymatic Digestion
This technique involves dewaxing, rehydrating, and
The blocks are then infiltrated and embedded with
rinsing the specimen in running water. The specimen
paraffin, and 5–10 μm thick sections are cut in ribbons
IHC / ICC

is then equilibrated with the appropriate buffer and is

and mounted on slides. Once mounted, the slides can
incubated with a proteolytic enzyme either at 37°C or
be stored indefinitely until immunostaining is required;
61
Antibody Applications

at room temperature. Enzymes used include pronase Autoclaving or pressure cooking is another method
(0.05% w/v in PBS), trypsin (0.05% w/v in PBS with 0.1% for HIER. In order to standardize the procedure, it is
CaCl2) and pepsin (0.05% w/v in 2N HCl). The conditions important to start with standard volumes of preheated
of concentration, time, and temperature must be solutions. After adding the specimens to the boiling
controlled, so that the enzymes can break some of the retrieval solution, the autoclave or pressure cooker
bonds formed during fixation, uncovering antigenic sites, should be brought to full pressure as quickly as possible
but the antigen should not be digested completely. The and the heating times measured exactly from this point.
enzymatic activity is stopped by placing the specimen At the end of the heating time (usually 1 to 2 minutes)
in cold buffer (4°C) prior to processing with antibody. the pressure should be released. As soon as possible
These methods should be considered for some antigens/ the hot buffer should be flushed out with cold water.
tissues. (Please see: Shi, S-R, et al. (1993). J. Histochem. (Sections should not be allowed to dry). The specimens
Cytochem. 41:1599–1604). However, proteolytic enzymes should then be washed in buffer. Although the most
can abolish the reactivity of some antigens (Please see: critical feature of both microwaving and autoclaving
Pileri, S., et al. (1997). J. Pathology 183: 116–123). is probably the heating of the tissues, the pH and
composition of the solutions used are also important in
Heat Induced Epitope Retrieval (HIER) the unmasking of antigenic sites. Studies have found no
HIER can be achieved through the following methods: significant difference between microwave and autoclave
• Microwave irradiation treatment, but there are significant differences based on
• Autoclaving or pressure cooking the solutions used. Some of the commonly used buffer
solutions are 0.01 M citrate buffer (pH 6.0), 0.1 M Tris-
Microwave irradiation of formalin-fixed, paraffin- HCl (pH 8.0), and 1 mM EDTA (pH 8.0), with citrate buffer
embedded specimens in buffer has been found to used most commonly. It should be noted that many
markedly enhance the retrieval of antigens. During this more specimens can be treated at any one time using
procedure, the energy provided helps break some of an autoclave or pressure cooker than in a microwave
the bonds formed during fixation, thus increasing the oven. However, preservation of the cytological detail may
number of available antigen-containing cells and the be slightly inferior in sections that undergo pressure
intensity of reactions. The exact mechanism, however, cooking. A recommended HIER protocol is available at:
is unclear. It is important to monitor tissue sections http://www.millipore.com/userguides/tech1/mcproto165.
during the microwaving process, to prevent damage
and drying. Maintaining consistent conditions between Citric acid incubation is a milder procedure that can be
experiments, including buffer volumes, irradiation times, used on many tissues. It is a simple incubation in citric
and microwave unit used, will result in less variability acid buffer, pH 3.0 for 30 minutes at 37°C after blocking,
in staining results. The number of samples that can but prior to primary antibody addition. Rinse slide in PBS
be treated by microwave irradiation at one time is or TBS, pH 7.4, prior to staining. The buffer composition
limited. Typically, specimens in some buffer (see below) is 2.1 g citric acid added to 400 mL of ddH2O. pH is
are heated either at full or partial power for a few adjusted to 3.0 with acetic acid if above 3.0, or with 1N
minutes. Periodically the heating is stopped and liquid is NaOH if below 3.0, make up to 1 L final volume with
replenished. After a set time, the solution containing the ddH2O.
slides is allowed to gradually cool to room temperature;
the slides are then rinsed in PBS and used for staining.
IHC / ICC

62
 Antibody Applications
3.7.4 Antibody Staining
There are several ways to exploit the specificity of antibodies to visually localize protein and other targets of interest in
tissue sections or cultured cells.

Direct labeling uses a primary antibody directly conjugated to an enzyme or fluorophore.

Indirect labeling is a two-step process requiring a primary antibody and secondary antibody, which is specific to the
light or heavy chain (or both) of the primary antibody and is conjugated to an enzyme or fluorophore.

The signal created by either the direct or indirect labeling method can be enhanced or amplified using biotinylated
primary or secondary antibody, which will bind multiple streptavidin proteins conjugated to an enzyme or fluorophore.

Immunohistochemistry Process

Direct Labeling

Figure 17.
IHC processes include direct
labeling (top), indirect
labeling (middle) and
indirect labeling with signal
amplification (bottom).
Add Conjugated 1° Ab

Indirect Labeling

Add Unconjugated 1° Ab Add Conjugated 2° Ab

Indirect Labeling with Signal Amplification

Add 1° Ab Add Biotinylated 2° Ab Add Amplifying Streptavidin

Visualizing enzyme-conjugated immune complexes involves an additional step in which a chromogen is catalyzed
by the enzymes to produce a colored pigment or dye. Horseradish peroxidase (HRP) and alkaline phosphatase (AP)
are typical enzyme conjugates, but the list of compatible chromogens is constantly growing. Proper microscopy
techniques and equipment are required to visualize the stained tissue or cells, while comprehensive knowledge of
histology will permit proper analysis.
IHC / ICC

63
Antibody Applications

The primary antibody may be directly labeled with an dilution will be that at which the strongest specific
enzyme (such as HRP or AP) or fluorophore (such as antigen staining is observed, with the lowest nonspecific
FITC or rhodamine), or it may be unlabeled, and require background. As with other controlled experiments, it is
detection by a labeled secondary antibody or a more advisable to change only one experimental variable at a
complex detection system. If a secondary antibody is time. After determining the optimum titer/dilution of the
used, it must be generated against the immunoglobulins primary antibody, the secondary antibody dilution can be
of the primary antibody source, e.g., if the primary optimized.
antibody is raised in rabbit, then the secondary antibody
could be goat anti-rabbit. The optimal titer of both the For staining of tissue sections, it is customary to
primary and secondary antibody should be determined incubate with 25–50 μL of diluted antibody (Note:
for each batch. The volume used must be sufficient to completely
cover the tissue, and to ensure the tissue will not dry
Streptavidin-peroxidase out during incubation). Incubation periods may range
Complex Peroxidase enzyme
Figure 18. oxidizes DAB, turning it from 30 to 90 minutes at 37°C, from one to six hours
Principle of colorimetric into a brown pigment
tissue localization of IHC at room temperature, or overnight at 4°C. Incubation
signals. times should be optimized empirically for each antibody/
This pigment
Antigen precipitates out of antigen combination.
of Interest solution as a brown solid,
located at the site

Nice to know
of our antigen.

TISSUE
Proper collection of animal tissue may involve
institutionally-approved euthanization or
The proper working dilutions for every antibody must be
anesthesia, perfusion, and appropriate gross
optimized for the system in which it is being employed.
dissection. Considerations when collecting
The same system does not always work for every
human tissue are: cause of death, medications,
antibody. The product data sheets may be used as a
morbidities, post mortem interval (PMI), and
guide for dilution series starting points. (See Appendix
handling times/conditions.
for a possible dilution protocol). The optimal antibody

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With increasing age, the autofluorescent pigment A. B. C.
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lipofuscin granules can complicate the use of
fluorescence microscopy in the central nervous
system because of its broad excitation and
Forty micron sections through the basal nucleus of Meynert from the brain of an adult human male. The
emission spectra, which overlap with those sections were untreated (A), treated with the Autofluorescence Eliminator Reagent (Cat. No. 2160) (B), or
of most commonly used fluorophores. The stained with a polyclonal goat anti-ChAT antibody (Cat. No. AB144P) and visualized with a Cy3-conjugated
secondary antibody followed by treatment with the Autofluorescence Eliminator Reagent (C). Images
Autofluorescence Eliminator Reagent will reduce were collected using a Bio-Rad Radiance 2100 MP Rainbow confocal microscope with a 20X objective.
or eliminate lipofuscin-like autofluorescence Illumination was provided by a 543 nm laser line, and emission was collected from 555-625 nm. The image
collection settings (laser power, PMT gain, pinhole diameter, and background level) were identical for all
without adversely affecting other fluorescent three images. Photos courtesy of Michael Hendrickson and Ronald Kalil, W.M. Keck Laboratory for Biological
label in sections of human, monkey or rat neural Imaging, University of Wisconsin-Madison.
IHC / ICC

tissue as well as other tissues.

64
 Antibody Applications
3.7.5 Antibody Detection outside of electron microscopy. The common antibody
Two of the most commonly used detection methods are: detection methods for light microscopy are described
• Colorimetric or Enzyme-mediated Detection right.
• Fluorescence-based Detection
Colorimetric or Enzyme-Mediated detection
With the advent of electron microscopy, detection of When choosing a substrate for conversion by an enzyme,
antigens by antibodies that contain large gold particles select a substrate that yields a precipitating product.
is often used as well. These may also be visualized at the Examples of commonly-used substrates are listed below.
light microscopic level, but their use is quite rare today,

Substrate Abbreviation Final Color Soluble in Alcohol (for counterstain)

Diaminobenzidine DAB Brown No

3,3'-diaminobenzidine (DAB) produces a brown end product which is highly insoluble in alcohol and other organic solvents.
Oxidation of DAB also causes polymerization. DAB has the ability to react with osmium tetroxide, and thus is very useful in
electromicroscopy as well as traditional immunohistochemistry sections.
Diaminobenzidine with DAB/Nickel Gray/Black No
nickel enhancement
Horseeradish Peroxidase

DAB/Nickel produces a more intense stain which is resistant to alcohol and provides better contrast, up to 40 times more
sensitive than DAB without enhancement.
3-Amino-9- AEC Red/Brown Yes
ethylcarbazole
3-Amino-9-ethylcarbazole (AEC) produces a red/brown reaction product and is widely used for immunohistochemical stain-
ing.. Slide specimens processed with AEC must not be immersed in alcohol or alcoholic solutions (e.g., Harris’ hematoxylin).
Instead, an aqueous counterstain and mounting medium should be used. AEC is also susceptible to further oxidation when
exposed to light and thus it will fade overtime. Dark storage and brief light viewing are recommended.
4-Chloro-1-naphthol 4-CN Blue/Gray Yes
4-chloro-1-naphthol (4-CN) precipitates as a blue end product. Because CN is soluble in alcohol and other organic solvents,
the slides must not be dehydrated, exposed to alcoholic counterstains, or coverslipped with mounting media containing
organic solvents. Unlike DAB, CN tends to diffuse from the site of precipitation, thus it is not usually recommended for Im-
munohistochemistry but can be used for Western blotting.
Naphthol AS B1 NABP/FR Red Yes
phosphate/fast red TR
Napthol AS acts as the substrate for alkaline phosphatase, and the Fast Red chromogen precipitates at the enzymatic sites
producing a vibrant red/pink color. Precipitate is soluable in alcohol, thus aqueous counterstain and mounting medium
should be used.
Naphthol AS MX NAMP/FR Red Yes
phosphate/fast red TR
Napthol AS acts as the substrate for alkaline phosphatase, and the Fast Red chromogen precipitates at the enzymatic sites
Alkaline Phosphatase

producing a vibrant red/pink color. Precipitate is soluable in alcohol, thus aqueous counterstain and mounting medium
should be used.
Naphthol AS B1 NABP/NF Red/Violet Yes
phosphate/new fuschin
Napthol AS acts as the substrate for alkaline phosphatase, and the new Fuchsin chromogen precipitates at the enzymatic
sites producing a vibrant red/violet color. Precipitate is soluable in alcohol, thus aqueous counterstain and mounting me-
dium should be used.
Bromochloroindolyphos- BCIP/NBT Purple No
phate/Nitro Blue
Tetrazolium
5-bromo,4-chloro,3-indolylphosphate (BCIP)/nitroblue tetrazolium (NBT) substrate is a commonly used substrate chromo-
gen. BCIP acts as the substrate for alkaline phosphatase, and the NBT enhances the purplish-brown color of the precipitate.
BCIP/NBT is compatible with organic solvents so it can be used with alcohol based counterstains including Nuclear Fast Red
or Methylene-Green.
IHC / ICC

65
Antibody Applications

Fluorescence-based detection
A molecule that fluoresces can be attached to the antibody for detection using UV light. Examples are fluorescein,
rhodamine, Texas Red®, Cy3, and Cy5. In selecting fluorochromes, one is limited by the available microscope filter sets.
Most filter sets are best matched with rhodamine or fluorescein. Texas Red® may also be used with a rhodamine filter set.

Many mounting media Fluorophore Absorption (nm) Emission (nm)

Ultraviolet
contain “anti-fading”
solutions, such as DABCO, Fast Blue 360 440
which will prolong the
viewing time of the Alexa Fluor® 350 346 445
sample. EMD Millipore AMCA 350 450
offers a variety of
fluorescence mounting Bisbenzamide 360 461
390
fluids and counterstain Aequorin Ca2+ photoprotein 469
solutions including our 400
basic fluorescent mounting Hoechst 33258 360 470
fluid (Cat. No. 5013) and
ACMA UV 0 412, 43 471, 474 410
enhanced counterstaining
fluid containing nuclear Hoechst 33342 343 483 420
stains such as 4’,
6-Diamino-2-phenylindole Cy2 489 506 430
dihydrochloride (DAPI; GFP Wild type Non UV ex. 475 509
Cat. No. S7113). 440
GFP Wild type UV ex. 395 509
450
Alexa Fluor® 488 494 517
460
Calcein 496 517
Fluorescein (FITC/DTAF) 495 520 470
Fluoro-Jade® B 480 525 480
Lucifer yellow 425 528 490
JC-1 514 529 500
Fluoro-Gold™ (Hydroxystilbamidine) 361 536
510
Alexa Fluor® 430 430 545
520
Eosin 524 545
6-JOE UV 520 548 530
Alexa Fluor® 532 530 555 540
Cy3 548 562 550
Alexa Fluor® 546 554 570 560
Alexa Fluor® 555 555 571
570
TRITC 547 572
580
B-phycoerythrin 545, 565 575
R-phycoerythrin 480, 545, 565 578 590
Rhodamine 539, 574 602 600
Alexa Fluor® 568 578 602 610
Texas Red® 589 615 620
Alexa Fluor® 594 590 617
630
Propidium Iodide (PI) 536 617
640
Ethidium Bromide 493 620
Feulgen (Pararosanoline) 570 625 650
Acid Fuchsin 540 630 660
Alexa Fluor® 633 621 639 670
Alexa Fluor® 647 649 666 680
Cy5 650 670
690
PE-Cy5 conjugates 480, 565, 650 670
700
Alexa Fluor® 660 668 698
Alexa Fluor® 680 684 707 710
Infrared
IHC / ICC

PE-Cy7 conjugates 480, 565, 743 767

767
Cy7 743 767

66
 Antibody Applications
3.7.6 Counterstaining in and nuclear Fast Red) and fluorescent dyes (e.g., DAPI and
Immunohistochemistry phalloidin) are commonly used to fit the experimental
During the past few decades, histochemical staining has design.
largely been replaced by immunostaining techniques.
However, a limited number of available colors is a major Hematoxylin is one of the most commonly used dyes for
drawback in the microscopic examination of tissues. nuclear staining. Oxidized hematoxylin is combined with
Hence, histochemical counterstaining techniques are aluminum ions to form an active metal-dye complex,
employed to overcome this difficulty. Double-staining which provides a blue color to the nuclei in mammalian
and even triple-staining of tissue slices offer several cells by binding to lysine residues on histones. On the
advantages and allows for the examination and other hand, nuclear Fast Red stains nucleic acids and is
identification of tissue morphology and sub-cellular much faster than hematoxylin. Fluorescent stains, such
location of antigens present. A second stain is applied as DAPI and Hoechst, intercalate into the DNA to give a
to provide contrast and allow the primary stain to stand strong blue color under ultraviolet excitation.
out. In this regard, both chromogenic (e.g., hematoxylin

3.7.7 Troubleshooting:
Problem Possible Cause Possible Solution
No staining No antigen Check literature to see if the protein is expressed in that particular tissue
type. Or, check mRNA expression by in situ hybridization.
Improper storage of antibodies Aliquot and store antibodies in smaller volumes. Store as recommended by
the manufacturer. Avoid repeated freeze-thaw cycles.
Insufficient tissue fixation Try a fixative or change the duration of fixation.
Overfixed tissue Reduce duration of fixation or fix tissue at +4°C instead of room temperature.
Primary and secondary Use secondary antibody that will interact with the primary antibody (see
antibodies not compatible section 2.5 for use of secondary antibodies).
Defective secondary reagents Prepare fresh reagents.
Enzyme-substrate reactivity; Deionized water may contain peroxidase inhibitors that can reduce enzyme
improper pH of substrate buffer activity. Use buffer in the recommended pH range for specific substrates.
Overstained Primary and/or secondary Determine optimal concentration, try lower concentrations.
Tissue antibody concentration is too
high.
Incubation period is too long. Try shorter incubation. Optimize the duration with each component:
antibody, substrate, enzyme etc.
Nonspecific binding of primary Treat tissues to minimize or block nonspecific binding.
and/or secondary reagents to
tissues
High Tissues may have high levels Try incubating with normal serum from species other than the source of
Background of endogenous molecules that primary antibody.
are also present in incubation
For tissues containing interfering peroxidase: treat tissue with 0.3%
mixtures. For example,
hydrogen peroxide in methanol for 30 minutes at room temperature.
peroxidase in blood cells may
remain in tissues.
Secondary antibody cross- The secondary antibody may show a strong or moderate affinity for identical
reactivity or nonspecific binding or similar epitopes on non-target antigens. For example, egg whites,
sometimes used to coat slides, contain high amounts of avidin. Avoid egg
whites to prevent avidin from binding biotinylated secondary antibody during
staining.
IHC / ICC

67
Antibody Applications

3.8 Flow Cytometry

3.8.1 Introduction
3.8.2 Antibodies in Flow Cytometry Laser Excitation Detectors
Green Fluorescence
3.8.3 Detection Methods Orange Fluorescence
3.8.4 Sample preparation Size/Refracted Light

Key Steps
3.8.5 Blocking
3.8.6 Antibody Incubation Cell Sorting
3.8.7 Data Acquisition
3.8.8 Troubleshooting Green Fluorescence Orange Fluorescence
Untagged

3.8.1 Introduction Figure 19.

Flow cytometry is a statistically powerful technique for Flow cytometers, such as the guava easyCyte™ 8HT
instrument, use laser excitation of single cells and
characterizing and/or sorting heterogeneous, suspended subsequent detection of fluorescence and refracted light to
cell populations on the basis of physical characteristics provide multiparameter cellular analysis.
and fluorescence. The prototype flow cytometer was
developed over 50 years ago; the technology was not
first commercialized until the late 1960s. The Cytometry Frontier
The last decade has witnessed widespread
Flow cytometry is defined as the measurement of the adoption of microcapillary flow cytometry,
cellular and fluorescent properties of particles (such as which uses smaller sample volumes and less
cells) in liquid suspension as they pass by a laser or other reagents, generates less waste and has lower
light source. operating costs than traditional, sheath
fluid-based flow cytometers. Flow cytometry
From these measurements, specific populations and technology has also been further miniaturized
subsets within them are defined and can even be into ultra-compact, benchtop cell analyzers.
physically isolated via cell sorting, where cell charge Together, these benchtop instruments have
is manipulated based on fluorescence characteristics transformed flow cytometry into nearly routine
to allow electrostatic deflection of particles. Adherent step in most cell-based analyses.
cells and solid tissues may also be analyzed if they
Imaging flow cytometry combines the
can be successfully dissociated to create a single-cell
statistical power and high speed throughput
suspension.
of flow cytometry with the visualization
capabilities of immunocytochemistry and
Flow cytometry relies on hydrodynamic focusing of a cell
microscopy. This combination yields a more
suspension sample to create a single-cell stream which
comprehensive set of protein localization and
passes in front of a laser. The manner in which the cell
distribution data from a single cell sample than
scatters incident light is used to determine the size and
any single technology introduced to date.
intracellular complexity of cells at a rate of thousands of
particles per second. This analog data is then converted More information on these cellular analysis
to digital data which can be quantified and plotted in platforms is available at:
two (or three) dimensions. www.emdmillipore.com/cellularanalysis
Flow Cytometry

68
 Antibody Applications
conjugates, because species cross-reactivity and binding
Flow cytometry in the clinic: of secondary antibody to unintended targets can easily
render data uninterpretable.
No well-equipped modern clinical laboratory
is complete without a flow cytometer, which
3.8.3 Detection Methods
has become a fundamental medical screening
As with other immunodetection applications, there are
and diagnostic tool. Clinical laboratory
several approaches to the use of antibodies to probe for
scientists are certified to perform flow
specific cell moieties by flow cytometry. There are three
cytometric analysis of peripheral blood samples
main methods:
whenever the physician orders a complete
• Direct Detection
blood count (CBC), a test which historically
• Indirect Detection
required microscopic examination of a blood
• Biotinylated Detection
smear. Therefore, this analysis was previously
statistically limited to the number of fields it
Direct Detection
was feasible for the pathologist to examine. For
Direct detection refers to a single-step staining process
CBC, the differential light-scattering properties
that employs a primary antibody that specifically binds to
of leukocytes are exploited to make rapid and
an epitope of interest and that is directly conjugated to a
statistically reliable determinations regarding
molecule that permits visualization or other detection of
relative abundance of peripheral blood cell
the binding event.
types. Forward and side scatter can even aid
in detecting size and shape abnormalities
When probing for antigens localized to the cell surface,
arising from conditions such as anemia and
fixation of cells is not recommended, as this process
myelodysplastic syndromes. The addition of
renders antigens of interest inaccessible to antibody
antibody testing for known disease markers
probes. It is therefore necessary to work efficiently
increases the value of flow cytometry in
to keep unfixed cells viable until data acquisition is
clinical diagnosis and assessment of treatment
complete. Use of these primary conjugates expedites the
progress.
staining process, as binding of the antigen of interest
and labeling with the detection fluorophore are achieved
in a single step.
3.8.2 Antibodies and Flow Cytometry
Although cell populations may be broadly characterized Indirect Detection
by light-scattering properties, more precise identification In the indirect detection method, incubation with a
of subpopulations requires use of probes which bind to purified antibody to permit binding to antigen of interest
specific surface or intracellular moieties unique to cell is followed by a fluorophore-conjugated secondary
subtypes. For example, all lymphocytes in a peripheral antibody specific for the primary antibody host isotype,
blood sample may be of the same size and intracellular forming a primary-fluorescent secondary antibody
complexity. Fluorescently-conjugated antibodies specific scaffold. Increased modularity in an antibody library may
for cell surface receptors (such as CD4, CD8, and CD19, be achieved by use of purified primary antibodies and
for example) can be applied to the cell suspension to fluorophore-conjugated secondary antibodies in a variety
identify helper T cells, cytotoxic T cells, and B cells, of wavelengths (or ‘colors’) and specific for host isotypes
respectively. in which the primaries are raised.

At a minimum, even basic, single-laser flow cytometers

are typically equipped with sufficient filters to permit Watch Out
detection of four fluorophores simultaneously, and Internalization of receptors or other surface
it is currently possible to differentiate as many as 18 antigens of interest occurs naturally when
wavelengths of light in a single experiment. Acquisition cells are removed from in vivo tissues or in vitro
Flow Cytometry

of signal from these complex fluorophore palettes culture conditions. To minimize this, unfixed
requires multiple lasers, appropriate filters, and an cells must be kept on ice and/or at 4°C.
antibody selection that consists principally of primary

69
Antibody Applications

80 120 160 200

Biotinylated Detection
A third alternative, biotinylated detection, exploits the
avidins, bacteria-derived proteins so named for their

Count
natural avidity for the endogenous ligand biotin. The
biotin-streptavidin complex is sometimes referred

0 20 40
to as ‘molecular velcro’, as its use as a research tool
to bind two molecules has become widespread in 100 101 102 103 104
Yellow Fluorescence
immunodetection, proteomics, affinity purification, and
analysis of nucleic acid-protein interactions. A diverse
selection of biotinylated primary or secondary antibodies Figure 20.
Staining of human peripheral blood mononuclear cells
is widely available commercially; flow cytometric
(PBMC) using a T10B9 primary Milli-Mark™ anti-CD3-
detection is achieved upon subsequent incubation PE antibody (yellow histogram, Cat. No. FCMAB168P).
Unstained PBMCs are shown as a gray histogram. Cells were
with streptavidin conjugated to fluorophore. Signal analyzed using a guava easyCyte™ 8HT flow cytometer.
amplification may occur because biotin has four sites
to which streptavidin may bind, resulting in a potential
fourfold increase in fluorescent signal for each primary
binding event.

Watch Out
Unfixed cells are fragile and it does not take much for them to be destroyed. To keep unfixed cells
alive until acquisition on the flow cytometer, it is critical to employ steps that are gentle, fast, and
efficient.

Advantages Disadvantages
Binding of antigen of interest and detection of bound Multicolor palette versatility is limited by the selection of
antibody are achieved in a single incubation, saving antibodies in different ‘colors’ that is financially feasible to
Direct Detection

time and allowing for maintenance of cell viability. acquire and store.
Cross-reactivity and unintended binding of antibody
are limited, allowing choice from a wider selection
of host antibodies (for example, a primary conjugate
raised in mouse may be used to detect antigen in a
sample of mouse cells).
Increased modularity in multicolor ‘channel’ selection Two incubation steps are required, mandating increased
with fewer antibodies on hand efficiency to avoid placing unfixed cells at risk for apoptosis
before data acquisition can be completed.
Indirect Detection

Potential for nonspecific binding by the secondary antibody is

introduced, necessitating incorporation of negative ‘isotype
controls’ for each host and antibody isotype used.
Care must be taken when selecting antibodies to ensure that
primary antibody is not raised in the same species from which
the sample is collected, as application of the secondary will
result in binding of both the primary and of endogenous
targets.
Potential for increase in fluorescent signal for each Increased risk for false-positive signal arising from avidin-
Biotinylated Detection

primary antibody:target antigen binding event fluorophore complex binding to endogenous biotin present in
the biological sample. Blocking of endogenous biotin may be
necessary depending on the nature of cells in the sample.
Flow Cytometry

70
 Antibody Applications
Key steps in flow cytometry include: For tube-based flow cytometry, sample titers of 0.5 – 1
• Sample preparation x 106 cells per sample will yield a single cell suspension
• Blocking when resuspended in 350 – 500 µL of buffer. Appropriate
• Antibody Incubation sample aliquots for high-throughput cytometers that use
• Data Acquisition multiwell plates are usually in the range of 1 x105 – 0.5
x 106 viable cells per well. If using a microcapillary flow
cytometer, such as the guava easyCyte™ flow cytometers,
3.8.4 Sample Preparation use approximately 10,000-100,000 cells per tube or plate
Any cells which can be made into a single cell suspension
well.
can be assessed by flow cytometry.
• To prepare an ex vivo cell population, freshly dissected
Following each manipulation of the cell sample, the flow
tissue is often gently homogenized using mechanical
cytometry tube/plate well should be filled with wash
dissociation methods, and different cell types are
buffer and gently agitated/vortexed to wash the cells
separated via density gradient centrifugation to
before re-pelleting.
remove intracellular matrix material, debris, and
irrelevant cell populations.
Unfixed cells should generally be centrifuged at
• Adherent cell lines must be detached from cell culture
300-500 x g; fixed cells are slightly more hardy, but
vessel surfaces using enzymatic solutions or calcium-
this force should be sufficient to pellet both fresh and
chelation reagents.
fixed samples. A five minute spin is usually sufficient
• Suspended cell cultures only need to be counted and
to create a secure pellet that will permit rapid and
assessed for viability.
efficient decanting by flicking the tube or tapping the
inverted multiwell plate against an absorbent pad. To
Cell counts/titers refer to viable cells in suspension; this
resuspend the pellet, rake the tube or well bottom a
can be determined either by using an automated cell
few times across a peg rack. This quickly frees the pellet
counter, such as the Scepter™ handheld cell counter
for resuspension before the next incubation, wash or
or the Muse® cell analyzer, and applying gating to
acquisition step.
exclude dead cells/debris. Alternatively, viability can be
determined by microscope-aided counting of the number
3.8.5 Blocking
of cells in a known volume (such as that provided by
To prevent nonspecific binding of primary antibody(ies)
a hemacytometer) in the presence of Trypan blue dye,
to suspended cells, an anti-Fc antibody dilution (specific
which is excluded by the intact membrane of live cells, so
to the sample species) may be applied. This prevents
that nonviable cells are easily identified by their uptake
binding of the Fc or constant region of the antibody
of the dye.
by Fc receptors which are present on most cell types.
Fc block is typically added to washed cells in extremely

Tech Tip small (~10 μL) volume as the staining antibody dilution is
added immediately at the end of the blocking incubation
Use nonenzymatic methods for detaching without a wash step. This ensures that blocking of
adherent cells from culture surfaces whenever nonspecific antibody binding is maintained throughout
possible to avoid unintentional cleaving of
the primary incubation.
antigen from cell surfaces. When adherent
cells are not amenable to detachment by non-
enzymatic means, it is essential to use enzymes
that are selective for attachment proteins (such
Tech Tip
as Accutase™ enzyme, Cat. No. SCR005), rather For a specific signal, and to minimize nonspecific
than general proteases such as trypsin. binding of the secondary to cells in the sample,
pre-block the cells and include 1 – 5% serum
(from the species in which the secondary
antibody was raised) in the staining buffer.
Flow Cytometry

71
Antibody Applications

3.8.6 Antibody Incubation secondary in order to permit differentiation of signal

Primary incubation from each target. Incubation with secondary antibody for
Unlike other antibody-based applications such as 20-30 minutes is carried out in the dark to protect light-
immunohistochemistry, dilution of antibody for flow sensitive fluorophores and, as before, samples should be
cytometry is typically based not on mass of antibody maintained on ice and centrifuged at 4°C.
per volume of buffer, but on mass of antibody per
number of cells in the sample. As with other applications, Streptavidin incubation (if needed)
optimal concentration must be empirically determined, If the available primary or secondary is biotinylated,
but typical dilutions are between 1 – 3 µg of antibody the final incubation step will be with an avidin, usually
per 10 cells for both primary and secondary antibody.
6 streptavidin, linked to a fluorophore. Incubation for 15-
Antibody may be diluted in flow cytometry assay buffer. 30 minutes on ice in dark conditions is followed by three
Staining in very small (50 - 100 µL) volumes improves washes to removed unbound streptavidin-fluorophore.
access of antibody to cells in suspension. At the end of Sample is then resuspended for in an appropriate volume
the incubation period, cells should be washed in staining of assay buffer, usually 200 - 500 μL per sample, and
or assay buffer three times to remove any unbound protected from light and maintained on ice until data
primary antibody. aquisition.

Fluorochromes
Tech Tip Many antibodies used in flow cytometry are directly
conjugated to a fluorochrome; however, many
If primary antibody is conjugated to a
unlabeled primary antibodies are routinely used in
fluorophore, the antibody incubation step must
be carried out in the dark to prevent loss of combination with labeled secondary antibodies. Two
signal via bleaching. common fluorochromes used in flow Cytometry are
fluoroisothiocyanate (FITC) and phycoerythrin (PE).
The two key properties of these dyes that make them
Secondary incubation preferred tags are that they are both excited with a 488
If indirect detection is employed, primary incubation nm laser and that their emission spectra are distinct,
is followed by incubation with appropriate dilutions with FITC at 530 nm (green) and PE at 570 – 575 nm
of secondary antibody specific for the isotype of (orange). Advances in fluorochrome chemistry and in
each primary antibody used. In multicolor detection flow cytometry instrumentation have made multiple
experiments, fluorophores of sufficiently different simultaneous cell labeling and sorting possible beyond
wavelengths or colors must be chosen for each the original two dyes.

Alexa Fluor® 488 100

Alexa Fluor® 750
Relative Intensity (%)

Cy3
Cy5.5-Allophycocyanin 75
Rhodamine Red
Fluorescein (FITC) 50
Hoeschst 33258

25

0
300 400 500 600 700 800 900
Wavelength (nm)

Figure 21.
Flow Cytometry

Diverse fluorochromes with nonoverlapping emission spectra enable multiparameter flow cytometry.

72
 Antibody Applications
Fixation and Storage
Proper Controls for Flow Cytometry Once surface antigen staining has been completed,
cells may be fixed in 3-4% paraformaldehyde in
In addition to cells of interest, every flow
phosphate-buffered saline instead of resuspending
cytometry experiment should include the
for acquisition. This is useful when samples cannot be
following controls:
acquired immediately after staining, as it allows cells
1. At least one unstained sample that has to be stored overnight at 4°C. Afterwards, the fixative
been incubated with buffer at each step should be diluted and the cells washed two times.
at the same time as the test samples. Although acquisition immediately following staining is
Unstained samples will be needed at the recommended, fixed cells that have been stored at 4°C
time of acquisition to optimize flow rates, away from light may be acquired up to 48 hours after
set detector voltages for forward and side fixation.
scatter, and to establish baseline voltages
for each fluorescence detector used in the Intracellular targets: permeabilization
experiment. When the protein(s) of interest are intracellular, fixation
of cells after surface staining is necessary to increase
2. An appropriate negative control sample
structural strength so that the cells can withstand the
which will be identical to the test samples
subsequent permeabilization needed to allow antibodies
except for substitution of the primary
access to intracellular antigen. Cells that have been fixed
antibody with an isotype control which
and washed, as described above, may be permeabilized
is raised in the same host species as the
by incubation with a 0.5% detergent in phosphate-
primary antibody. This control is necessary
buffered saline for no more than 15 minutes at room
to establish whether any of the fluorescent
temperature before diluting detergent solution and
signal detected is due to direct nonspecific
washing once. Nonionic detergents such as saponin are
binding of the secondary antibody to the
recommended. Permeabilization must be maintained
sample cells, and permits subtraction of
during all steps involving antibodies or streptavidin,
this ‘background’ from the fluorescence
and this is achieved by including 0.1% detergent in the
signal. An alternative, less robust negative
staining buffer throughout subsequent staining up to
control is a sample identical to the test
and including the fluorophore incubation step. Detection
samples from which the primary antibody
of intracellular antigen by flow cytometry otherwise
has been omitted.
follows the principles and procedures outlined above
3. Where possible, a positive control which is and may be direct, indirect, or direct/indirect with signal
comprised of cells known to express each enhancement.
antigen of interest and is co-incubated
with the test samples. For multichannel
experiments, each wavelength channel to Tech Tips
be used must have a corresponding single-
Surface and intracellular antigen staining are
color positive control sample incubated
frequently combined to permit characterization
with the same antibody(ies) as the test of subsets of cells; a sample experiment might
samples. Control beads are usually sold for be as follows:
this purpose, and should be used according
• Collect whole blood and isolate PBMCs by
to the manufacturers’ instructions.
density centrifugation.
However, cells may be used as single-color
positive controls provided that they are • Treat PBMCs with a compound suspected to
certain to express the target antigen. be genotoxic.

• Stain for surface markers such as CD4, CD8,

CD19, NK1.1.
Flow Cytometry

• Fix and stain with anti−γH2.AX antibody to

assess induction of double strand breaks in
lymphocyte DNA.

73
Antibody Applications

3.8.7 Data Acquisition When one wishes to compare multiple parameters that
Flow cytometers are accompanied by the software are collected at the same time, more complex two-
necessary to acquire and transform the signals generated dimensional and even 3-dimensional diagrams are
by the particle characteristics as each cell passes by the required. In these diagrams, one parameter is plotted
detector. These software programs usually also include against another in an X versus Y axis display (Figure 23).
components that aid in organizing the experiment, a
feature that is particular important when analyzing a

9000
variety of samples from multiple specimens or donors.

7000
Once fluidics are established, unstained cells can be used

Side Scatter
to set forward and side scatter detectors, and fluorescent

5000
detectors adjusted by comparison of unstained cells

3000
to positive controls. The user also sets the software
to collect a uniform number of ‘events’, or particle/cell

0 1000
signals, for each sample. For multicolor experiments, 0 1000 3000 5000 7000 9000
it is also critical to set parameters for compensation, Forward Scatter

in recognition of the wavelength overlap amongst

spectra of the fluorophores used in the experiment. Figure 23.
Compensation calibrates each fluorophore’s spectrum The two-parameter flow cytometry plot at left shows how
light-scattering characteristics of cells are used to identify
and allows for subtraction of signal from adjacent cell populations of interest, here by their size (forward
channels due to spectral overlap. scatter) and intracellular complexity (side scatter). Gates,
or regions defined and applied by the user, are then used
to identify subpopulations of interest (here, the red oval
Single-parameter histograms display the relative identifies the likely viable population) for focused analysis.

fluorescence plotted against the number of events. The

simplicity of this type of display is the main reason for
Labeling of cells in flow cytometry is often done using
its popularity. Its ease of use makes it ideal for simple
antibodies specific to particular subclasses of cells.
assays, for instance distinguishing apoptotic cells from
Antibodies typically used are specific to external cell
non-apoptotic cells (Figure 22).
epitopes for live cell sorting. Internal epitope antibodies
are typically only used on fixed cells that have first been
60

permeabilized to allow the antibody penetration. EMD

Millipore offers numerous polyclonal and monoclonal
50

antibodies specific to different cell types. Many of the

Count
40

groups of cell-type specific monoclonals have been given

a cluster of differentiation (CD) number by international
20

convention. For example, the monoclonal antibodies

0

100 101 102 103 104 recognizing epitopes of the antigen site on helper T cells
PM1 Fluorescence
are known collectively as CD4 (Figure 24).
100

Figure 22.
Flow cytometry data of HL-60 cells that were treated with
80

apoptosis inducer camptothecin and subsequently fixed and

stained with the Guava® TUNEL apoptosis assay (Cat. No.
40 60
Count

4500-0121). Approximately half of the cells are apoptotic

(green histogram).
20
0

100 101 102 103 104

Anti-Human CD4-FITC

Figure 24.
Flow Cytometry

Flow cytometry analysis of human lymphocytes stained

with anti-human CD4-FITC antibody. Cells expressing CD4
(right hand peak) were distinguished from CD4-negative
cells (left). CD marker expression can vary between different
blood specimens.

74
 Antibody Applications
Tech Tips
• Mix samples thoroughly before use; avoid excessive bubbling.
• If minor precipitate is detected in the wash buffer, place the bottle in a warm water bath for 30 minutes,
followed by mixing the contents on a mechanical vortexer.
• For cellular staining and analysis to be most effective, make sure that test cells have good viability prior
to use.
• For certain cell cultures, cell pellets may become hazy or transparent following the fixation step, making
them difficult to see. If sampling a small collection of cells for flow analysis, it is recommended to
perform all steps in a smaller collection tube (e.g. microcentrifuge tube).
• Do not mix or interchange reagents from various kit lots.

3.8.8 Troubleshooting:
Problem Cause Suggested Solution
Acquisition Blocked fluid Decrease the number of cells being analyzed by diluting the sample to approximately 0.5 million cells per mL. Cell
rate decreases pathway on the densities in excess can essentially block the normal flow, causing disruption during the assay.
dramatically instrument
After many uses, it is possible that the fluid system on any standard flow cytometer will require cleaning. Run standard
cleaning procedures to clean the fluid system during or after an assay. This will prevent any material from forming
where the steady flow steam takes place.
Cells are clumping Adherent or sticky cells can result in cellular clumping. Using a more aggressive enzyme for dissociation such as trypsin
during cell harvesting should help keep cells in single suspension. Alternatively, using a cell strainer can help disrupt
cell clumping (Cat. No. SCNY00060; 60 µM).
A loss or lack of Not enough cells Cell loss is common during washing steps in the assay procedure. Make sure that cell density remains at approximately
signal in the sample 0.5 million cells per mL during analysis.
Not enough A lack of signal may indicate that excess antibody will need to be used during the staining procedure. Further antibody
antibody titrations may be necessary for some cell types to capture the ideal staining concentration.
Background and/ Too much Nonspecific staining and background may indicate that less antibody will need to be used during the staining
or non-specific antibody procedure. Further antibody titrations may be necessary for some cell types to capture the ideal staining concentration.
staining of cells
Variability in Changes in cell Monitor experimental cell cultures to ensure that cell viability and cell numbers being analyzed are consistent.
results from day viability
to day
Instrument Perform quality check on the instrument (e.g. calibration) on a daily basis prior to use.
calibration
changes

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our imaging flow cytometers produce insightful
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Flow Cytometry

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your research needs and budget.

75
Antibody Applications

3.9 Functional Blocking and Stimulation Assays

Introduction
Specific antibody-antigen interactions may result in functional changes to the target protein. These interactions may
neutralize the antigen, as in the case of “neutralizing antibodies”, block or modulate an active site on an enzyme, or
bind and stimulate a conformational change in the protein. Whether an antibody will indeed operate in a functional
assay depends on the epitope of the antibody in relation to the 3D conformation of the protein domains and active
site. Thus, unlike the design of many small molecule inhibitors/activators, the design of an antibody to specifically
alter protein function is not easily predicted.

As with small molecules, antibodies may change protein function by:

1. Direct active site binding

Substrate
a. Reaction
Active Site

Enzyme

Substrate molecule binds with Reaction occurs and product

active site of enzyme molecule molecules are generated

Inhibitor/Antibody
b. Inhibition
Active Site

Enzyme

Inhibitor/Antibody molecule binds Inhibitor/Antibody molecule

with active site of enzyme molecule prevents the binding of substrate
molecule

2. Allosteric binding-induced conformational changes

a. Reaction
Substrate

Allosteric site Active Site

Enzyme

Substrate molecule binds with active site Reaction occurs and product
of enzyme molecule, including a change in molecules are generated
the allosteric site that prevents the
inhibitor from binding

b. Inhibition
Inhibitor

Active Site

Enzyme

Inhibitor molecule binds with Substrate molecule cannot bind

Functional Assays

allosteric site of enzyme, including with changed active site

a change in the active site

3. Steric hindrance
76 Substrate
a. Reaction
Allosteric site Active Site
 Antibody Applications
3. Steric hindrance
Substrate
a. Reaction
Allosteric site Active Site

Enzyme

Substrate molecule binds with active Reaction occurs and product

site of enzyme molecule, preventing molecules are generated
binding of inhibitor molecule
b. Inhibition

Inhibitor
Active Site

Enzyme

Inhibitor molecule binds with active Inhibitor molecule prevents the

site of enzyme molecule binding of substrate molecule

Unlike many small molecules, however, antibodies have Neutralizing antibodies are also important in the
poor cell membrane penetration and thus are restricted characterization of the safety and efficacy of large
to external epitopes in live cell functional assays. The molecule biotherapeutics, which may elicit anti-drug
non-penetrance but specificity of antibodies is a great antibody (ADA) responses in the host. Regulatory
advantage in targeted blocking or stimulating of cell agencies have issued biotherapeutic testing guidelines,
surface proteins. Many antibodies can be designed to which recommend performing functional assays for the
strongly antigenic epitopes at or near binding sites, presence of neutralizing antibodies in nonclinical and
resulting in steric inhibition of receptor-ligand binding. clinical studies. As a result, there has been a surge in the
These studies typically target channel or receptor development of novel assays for neutralizing antibodies.
deactivation or block protein-protein binding interfaces,
such as those occurring in cell adhesion. See the chart
below for examples. Watch Out
Many commercial antibodies have preservatives,
Some receptor binding antibodies are particularly such as sodium azide, which can poison live
useful in that the antibody-receptor complex is quickly cells. For live cell functional studies, it may be
internalized and degraded. These antibodies can be necessary to perform dialysis or diafiltration
conjugated to toxins such as Saporin, so when the on the resuspended antibody to remove the
complex is internalized, the toxin is released internally, preservative, or choose a preservative-free
killing the cell. This is a classic method for ablating version of the antibody from the manufacturer.

specific cell types in the brain. Cholinergic neurons, for

example, can be selectively eliminated targeting the
p75 NGF receptor with a corresponding antibody-toxin
conjugate.

Function Description Reference Antibody example Cat. No.

Immunolesioning Induces cholinergic cell death Wiley, R. (2001). Methods Mol. Biol. 169:217. Anti-p75 MAB390-25UG
NGFR-Saporin conj.
Enzyme function blocking Inhibits MMP-2 activation Chen, H. et al. (2012). J. Biol. Chem. 287:17109. Anti-MMP-2 Proform MAB13405
Receptor blocking Inhibits TLR2 activation Sandor, F. et al. (2003). J. Cell Biol. 162:1099 Anti-TLR2 MAB3737
Blocking cell adhesion Blocks aVb3 complex binding Mao, Y. & Schwarzbauer, J. (2005). J. Cell Sci. 118:4427. Anti-aVb3 MAB1976Z
to substrate to ECM
Functional Assays

Blocking cell-cell adhesion Blocks integrin b1 / CD63 Iizuka, S. et al. (2011). Mol. Cell Biol. Anti-Integrin b1 MAB2253Z
interaction
Neutralizing ligands Neutralizes BDNF in vitro and Finn et al. (1986). J. Neurocytol. 15:169) Anti-BDNF AB1513P
in vivo
Stimulating receptor Induces apoptosis by receptor Trauth, B. et al. (1989). Science 245:301 Anti-Fas MAB3061
activation

77
Antibody Applications

Tech Tips
• Know your immunogen. Choosing the right antibody for function blocking depends on epitope, 3D
conformation of the active site of interest, and antibody binding avidity. For example, to interrupt
integrin aVb3 binding to substrates, choose an antibody that targets the binding site or allosterically
interferes with it. Such is the case of the well known mouse monoclonal antibody LM609. While
LM609 does block cell attachment to RGD containing ligands, it does not interact directly with the RGD
binding site. Instead, LM609 appears to be an allosteric inhibitor of integrin aVb3, which binds to a
conformational epitope resulting from the post-translational association of the aV and b3 subunits.
• Antibodies must be preservative-free in live cell assays. Use diafiltration- or dialysis-based buffer
exchange if necessary to remove preservatives.
• In vitro to in vivo protocol extrapolations can be difficult and typically include significantly increasing
the antibody concentration used. In vivo considerations also include prevalence of endogenous
immunogen, antibody delivery method, tissue architecture, and host immune response to foreign
antibody addition.
• In functional live cell assays, cell stimulation response should be at least five-fold over background
obtained at the EC50 antigen concentration to allow for a large enough signal due to potential variance
in antibody inhibition. Triplicate or quadruplicate trials are recommended.
• Antibody performance in a functional assay is more easily affected by changes in the medium
environment than is typical for nonfunctional immunoassays like IHC or ELISA.

Technology Highlight

Blocking adhesion with Mouse anti-Integrin aVb3

EMD Millipore has over 200 function blocking or stimulating anti-b1 anti-aVb5 anti-aVb3
antibodies. One example, Anti-Integrin αVb3 Antibody, clone LM609
reacts with the vitronectin receptor αVb3 complex, an RGD-directed
adhesion receptor. LM609 has been demonstrated to block adhesion
of a human melanoma cell line (M21) to vitronectin, fibrinogen
and von Willebrand factor, as well as to a synthetic RGD containing
peptide (Cheresh, 1987). In chick chorioallantoic membranes, LM609 Mouse anti-Integrin aVb3 (Cat. No. MAB1976). Inhibition of angiogenesis on the
chick chorioallantoic membrane by anti-aVb3. The pictures for MAB1976 and
was shown to block angiogenesis induced by bFGF and TNFα but had MAB1981 are reprinted with permission from Figures 1, 2, and 4 in: Brooks, P.C.,
Clark, R.A.F., and Cheresh, D.A. (1994) Science 264:569, © 1994 by the AAAS.
no effect on pre-existing vessels (Brooks, 1994). While LM609 does
block cell attachment to RGD-containing ligands, it does not interact
directly with the RGD binding site. Instead, LM609 appears to be an
allosteric inhibitor of integrin aVb3, which binds to a conformational
epitope resulting from the post-translational association of the aV
and b3 subunits.
Functional Assays

78
Notes

79
80
 SAMPLE PROTOCOLS
Sample Protocols 4
4.1 Immunoprecipitation 4.5 Multiplexed Bead-based Detection
4.2 Chromatin Immunoprecipitation (ChIP) 4.6 Immunohistochemistry/Immunocytochemistry
4.3 Western Blotting 4.7 Flow Cytometry
4.4 Enzyme-linked Immunosorbent Assays 4.8 Functional Blocking and Stimulation Assays
(ELISA)

4.1 Immunoprecipitation 5. Mix gently, and incubate on ice for 1 hour to allow
The protocol below details the steps for the immune complexes to form. Longer incubation
formation of the antibody-antigen complexes in the times may increase complex formation, but may also
immunoprecipitation procedure. To a microcentrifuge increase nonspecific background.
tube, add:
1. 500 μL of 2x Immunoprecipitation Buffer (1% Triton®
X-100, 300 mM NaCl, 20 mM Tris, pH 7.4, 2 mM EDTA,
Tips
2 mM EGTA, 0.4 mM sodium vanadate, 0.4 mM PMSF, • Antibody concentrations given are
0.5% NP-40) recommended starting points only. Optimal

2. 200–500 μg of total protein lysate (approximately antibody concentrations must be determined

100 μL of lysate at 2–5 mg/mL) empirically.
3. 1–5 μg of purified antibody. If unpurified antibodies • For the negative control reactions, add an
are used, substitute the following antibody quantities: equivalent amount of control antibody.
Serum: 0.5–5 μL
Hybridoma tissue culture supernatant: 10–100 μL
Ascites fluid: 0.1–1.0 μL
4. Add ddH2O to 1 mL.

4.2 Chromatin Immunoprecipitation 6. Add 2 mL of 10x glycine to each dish to quench

excess formaldehyde.
(ChIP)
Preparing cells for ChIP 7. Swirl to mix and incubate at room temperature for 5
minutes.
1. If necessary, stimulate or treat adherent mammalian
cells at ~80 to 90% confluence in a 150 mm culture 8. Place dishes on ice.
dish containing 20 mL of growth media. Include one 9. Aspirate medium, removing as much medium as
extra plate of cells to be used solely for estimation possible, being careful not to disturb the cells. If you
of cell number. are using suspension cells, spin down cells at 8000 x
2. Prepare 22 mL of 1x PBS (2.2 mL 10x PBS and 19.8 g for 5 minutes.
mL water) for each 150 mm culture dish. Store on 10. Add 10 mL of cold 1x PBS to wash cells. Remove 1x
ice. This will be used for washes and needs to be ice PBS.
cold.
11. Remove 1x PBS and repeat wash.
3. Add 550 µL of 37% formaldehyde (or 1100 µL of
12. Add 2 mL of 1x Protease Inhibitor Cocktail III in PBS
18.5% formaldehyde) directly to 20 mL of growth
prepared in Step 5.
media to crosslink. Gently swirl dish to mix.
13. Scrape cells from each dish into a separate
4. Incubate at room temperature for 10 minutes.
microcentrifuge tube.
5. During the ten minute incubation, prepare 1x
14. Spin at 800 x g at 4 °C for 5 minutes to pellet cells
protease inhibitor in PBS: Add 2 mL of ice cold 1x
PBS to a separate tube for every dish and add 10 μL
of the 200x Protease Inhibitor Cocktail III. For detailed protocols, tips and troubleshooting
Store on ice. strategies, consult EMD Millipore’s “Guide to Chromatin
Immunoprecipitation: Critical Factors for Success”
(Literature Number TP5994EN00) 81
SAMPLE PROTOCOLS

Preparing tissues for ChIP 2. Continue with the following Cell Lysis procedure. Each
1. Isolate unfixed fresh tissue as desired. Use a razor microcentrifuge tube should contain approximately
blade to cut a pea-size piece of tissue into small 500 μL of cell lysate.
pieces (typically 1 mm or smaller) to improve
crosslink efficiency. Alternatively, a plug of tissue
from cryosectioned non-formalin-fixed-paraffin- Volume of
Cell Lysis Buffer Cell Density Cells Required
embedded (non-FFPE) material can be used to
500 µL 5 x 10 /mL
6
2.5 x 106
obtain a small sample of interest (please see
the Magna ChIP® G Tissue Kit manual, Cat. No. 500 µL 2 x 107/mL 1 x 107
17-20000). 500 µL 5 x 10 /mL
7
2.5 x 107

2. Weigh the tissue, and then transfer into a 50 mL

tube and wash twice with ice cold 1x PBS. 3. Be sure to keep the samples on wet ice at all times.
3. Resuspend tissue in 20 mL ice cold PBS and add Sonication generates heat which will denature the
550 µL of 37% formaldehyde (or 1100 µL of 18.5% chromatin.
formaldehyde) to crosslink. Gently swirl dish to mix. 4. Remove 1 x 105 cell equivalents from each condition
4. Incubate at room temperature for 10 minutes. prior to sonication for analysis of unsheared DNA.

5. In the interim, prepare 1x protease inhibitor in PBS: 5. For each cell concentration, sonicate each tube for
Add 2 mL of ice-cold 1x PBS to a separate tube for a fixed number of cycles allowing rests between
every sample and add 10 µL of Protease Inhibitor cycles according to the instrument manufacturer’s
Cocktail III. Store on ice. guidelines. For example, using a Misonix 3000 instru-
ment and a No. 419 microtip probe, use six 15 second
6. Add 2 mL of 10x glycine to quench excess
pulses with 50 second intervals between pulses, with
formaldehyde.
power setting at 6. Keep tubes cool at all times.
7. Homogenize the tissues several times using a
6. Remove 1 x 105 cell equivalents (20 µL, 5 µL, 2 µL
Dounce homogenizer (loose pestle).
from least to most concentrated sample) of the soni-
8. Spin at 800 x g at 4 °C for 5 minutes to pellet cells. cated chromatin from each condition to a fresh tube.
7. To all samples (unsheared and sheared), add elution
Optimizing Sonication and Analyzing buffer to a final volume of 50 μL.
DNA Fragments 8. Add 1 μL Proteinase K and incubate at 62 °C for 2
Optimal conditions for shearing crosslinked DNA to hours.
200 - 1000 base pairs in length depend on the cell type, 9. Load 10 μL and 20 μL on a 1–2% agarose gel with a
cell concentration, and the specific sonicator equipment, 100 bp DNA marker. Loading different amounts helps
including the power settings and duration and number of to avoid under- or overloading
pulses. Approaches for optimizing sonication may include
the following: 10. Observe which of the shearing conditions gives a
smear of DNA in the range of 200 – 1000 bp.
A. Varying the concentration of cell equivalents per mL of
initial buffer with constant sonication parameters. 11. Repeat optimization of the shearing conditions if the
results indicate that the resulting DNA is not in the
B. Choosing a fixed concentration of cell equivalents per desired size range. Once optimal conditions have been
mL of buffer and varying cycles and/or power settings determined, it is advised that you do not alter the cell
of sonication. concentration or volume of lysate per microcentri-
C. A combination of both approaches. fuge tube for subsequent chromatin immunoprecipi-
tation experiments.

The protocol below describes optimization following

option A and is provided as an example only.
1. Generate a cell lysate by following the first section
above, but vary your buffer volume per cell
amount-to generate 3 different microcentrifuge tubes
containing several cell equivalent concentrations in
the range of 5 x 106 per mL to 5 x 107 per mL. For
HeLa cells, this requires approximately 4 x 107 cell
equivalents, or approximately four 15 cm plates.

82
 SAMPLE PROTOCOLS
DNA Purification 7. Normally, the aqueous phase forms the upper phase.
(The following protocol was adapted from Sambrook, et al., 2006.) However, if the aqueous phase is dense because of
salt (> 0.5 M) or sucrose (> 10%), it will form the
1. Centrifuge your samples at full speed for 5 min at RT.
lower phase. The organic phase is easily identifi-
2. Transfer supernatants to fresh microcentrifuge tubes. able because of the yellow color contributed by the
3. You can now purify your DNA using spin columns. For 8-hydroxyquinoline that is added to phenol during
solvent extraction, continue to Step 4. equilibration.
4. Transfer the nucleic acid sample to a polypropylene 8. Use a pipette to transfer the aqueous phase to a fresh
tube and add an equal volume of phenol:chloroform. tube. For small volumes (< 200 μL), use an automatic
The nucleic acid will tend to partition into the organic pipettor fitted with a disposable tip. Discard the
phase if the phenol has not been adequately equili- interface and organic phase.
brated to a pH of 7.8 - 8.0. 9. Repeat Steps 1–4 until no protein is visible at the
5. Mix the contents of the tube until an emulsion forms. interface of the organic and aqueous phases.
6. Centrifuge the mixture at 80% of the maximum 10. Add an equal volume of chloroform and repeat
speed that the tubes can bear for 1 minute at room Steps 2–4.
temperature. If the organic and aqueous phases are 11. Recover the nucleic acid by standard precipitation
not well separated, centrifuge again for a longer time. with ethanol.

4.3 Western Blotting

The standard Western blotting protocol involves Tips
immunodetection of blotted proteins directly after
• Immobilon®-PSQ transfer membrane has
electrotransfer. (If the membrane was dried after
a smaller pore size (0.2 μm) and higher
transfer, thoroughly wet the blot in methanol for 5
minutes before proceeding to immunodetection.) The surface area than Immobilon®-P ransfer
unoccupied membrane binding sites on the wet blot membrane (0.45 μm). Increased background
are blocked with optimized reagents. The drawbacks can be expected on Immobilon®-PSQ and
of this method are the need for blocking and the the blocking and wash steps will need to be
total time requirement of over 4 hours. For alternate,
adjusted accordingly.
time-saving immunodetection protocols, refer to
EMD Millipore’s Protein Blotting Handbook, Literature • Phosphatases in the blocking solution may
Number TP001EN00. The advantage is that standard dephosphorylate blotted proteins
immunodetection may require less optimization for new
• Do not use sodium azide in the buffers as it
sample types.
inhibits HRP activity.

Required Solutions • Do not let the blot dry out at any time
• Primary antibody (specific for protein of interest). during and after blocking.
• Secondary antibody (specific for primary antibody), • If more than one blot is placed in a
labeled with alkaline phosphatase or horseradish container, insufficient buffer volume will
peroxidase.
cause the blots to stick together.
• Substrate appropriate to the enzyme conjugate.
• Dry milk powder cannot be used with biotin-
• Phosphate-buffered saline (PBST): 10 mM sodium
avidin systems.
phosphate, pH 7.2, 0.9% (w/v) NaCl, up to 0.1%
Tween®-20 detergent. • Sensitivity of chromogenic detection is
• TBST: 10 mM Tris, pH 7.4, 0.9% (w/v) NaCl, up to 0.1% typically at least an order of magnitude
Tween®-20. lower than of chemiluminescent detection.
• Blocking solution: 1% (w/v) BSA (bovine serum • High nonspecific signal can be
albumin), 0.05% Tween®-20.
alleviated by higher dilution of the primary
• Milli-Q® water. antibody or reduced protein load on the gel.

83
SAMPLE PROTOCOLS

Chromogenic Detection
Tips 1. Prepare the substrate according to manufacturer’s
instructions.
• Immobilon®-FL membrane can be scanned
2. Place the blot in a clean container and add substrate
dry or wet.
to completely cover the surface of the membrane.
• High overall background can be minimized Incubate for 10 minutes with mild agitation or until
by higher dilution of the enzyme-conjugated signal reaches desired contrast.
secondary antibody. 3. Rinse the blot with Milli-Q® water to stop the reaction.
4. Store the blot out of direct light to minimize fading.
Blot may be stored dry.
Required Equipment
• Shallow trays, large enough to hold blot. Chemiluminescent Detection
• Glass plates. Follow manufacturer’s instructions.
• Plastic wrap (e.g., Saran™ film), freezer bag, or sheet 1. Prepare the substrate according to manufacturer’s
protector. instructions.
• Autoradiography film and cassette. 2. Place the blot in a container and add substrate to
completely cover the membrane. Incubate for 1 minute.
• Dark room.
3. Drain excess substrate.
• Autoradiography film processing equipment.
4. Place the blot on a clean piece of glass and wrap in
plastic wrap.
Set Up
1. Dilute the primary antibody in the blocking solution to Note: A cut-to-size sheet protector or a freezer bag

the desired working concentration. can also be used.
2. Dilute the secondary antibody in the blocking solution 5. Gently smooth out any air bubbles.
to the desired working concentration. Note: Enough 6. In a dark room, place the wrapped membrane in a film
solution should be prepared to allow for 0.1 mL of cassette.
antibody solution (primary and secondary) per cm2 of
7. Place a sheet of autoradiography film on top and close
membrane.
the cassette.
8. Expose film. Multiple exposures of 15 seconds to
Antibody Incubations
30 minutes should be done to determine the optimum
1. Place the blot in the blocking solution and incubate
exposure time; 1 to 5 minutes is common.
with agitation for 1 hour.
2. Place the blot in the primary antibody solution and
incubate with agitation for 1 hour. The solution should Fluorescent Detection
move freely across the surface of the membrane. Required Equipment
• Proteins blotted onto Immobilon®-FL transfer
3. Place the blot in PBS and wash for 10 minutes. Repeat
membrane and probed with antibodies
twice with fresh buffer.
• Mylar® wrap
4. Place the blot in the secondary antibody solution and • Fluorescent imaging equipment
incubate with agitation for 1 hour at RT or 37°C.
5. Place the blot in PBS and wash for 10 minutes. Repeat The following is a general protocol for fluorescent
twice with fresh buffer. immunodetection. For optimal results, refer to
manufacturer’s protocol provided with the reagents.
6. Proceed with either chromogenic, chemiluminescent
Note: If using chemifluorescent reagents, follow reagent
or fluorescent detection.
manufacturer’s directions.
1. Place the blot in diluted fluorescent dye-labeled
secondary antibody solution and incubate for 1 hour
with gentle agitation.
2. Wash the blot with wash buffer 3 -5 times for 5
minutes each.
3. Place the blot onto a piece of clean filter paper to dry.
4. If using a wrap, use Mylar. Do not use Saran™ wrap
because it permits light to shine through and quench
fluorescence.
5. Image the blot using an appropriate fluorescence
scanner.
84
 SAMPLE PROTOCOLS
4.4 Enzyme-linked Immunosorbent Note: Some enzyme substrates are considered
hazardous, due to their potential carcinogenicity.
Assays (ELISA) Handle with care and refer to Material Safety Data
Sandwich ELISA Sheets for proper handling precautions.
1. Before the assay, both antibody preparations should For quantitative results, compare signal of unknown
be purified and one must be labeled. samples against those of a standard curve. Standards
2. For most applications, a polyvinylchloride must be run with each assay to ensure accuracy.
(PVC) microtiter plate is best; however, consult
manufacturer guidelines to determine the most Competitive ELISA
appropriate type of plate for protein binding. 1. For most applications, a polyvinylchloride (PVC)
3. Bind the unlabeled antibody to the bottom of each microtiter plate is the best choice; however, consult
well by adding approximately 50 μL of antibody manufacturer guidelines to determine the most
solution to each well (20 μg/mL in PBS). PVC will bind appropriate type of plate for protein binding.
approximately 100 ng/well (300 ng/cm2). The amount 2. Add 50 μL of diluted primary antibody (capture
of antibody used will depend on the individual assay, antibody) to each well. The appropriate dilution
but if maximal binding is required, use at least 1 μg/ should be determined using a checkerboard titration
well. This is well above the capacity of the well, but prior to testing samples. PVC will bind approximately
the binding will occur more rapidly, and the binding 100 ng/well (300 ng/cm2). The amount of antibody
solution can be saved and used again. used will depend on the individual assay, but if
4. Incubate the plate overnight at 4°C to allow complete maximal binding is required, use at least 1 μg/well.
binding. This is well above the capacity of the well, but the
5. Wash the wells twice with PBS. A 500 mL squirt binding will occur more rapidly, and the binding
bottle is convenient for this. The antibody solution solution can be saved and used again. Allow to
washes can be removed by flicking the plate over a incubate for 4 hours at room temperature or 4°C
suitable container. overnight.

6. The remaining sites for protein binding on the Note: If a purified capture antibody is not available,
microtiter plate must be saturated by incubating the plate should first be coated with a purified
with blocking buffer. Fill the wells to the top with 3% secondary antibody directed against the host of
BSA/PBS with 0.02% sodium azide. Incubate for the capture antibody according to the following
2 hours to overnight in a humid atmosphere at room procedure:
temperature. A. Bind the unlabeled secondary antibody to the
Note: Sodium azide is an inhibitor or horseradish bottom of each well by adding approximately
peroxidase. Do not include sodium azide in buffers 50 μL of antibody solution to each well
or wash solutions if an HRP-labeled antibody will be (20 μg/mL in PBS).
used for detection. B. Incubate the plate overnight at 4°C to allow
7. Wash wells twice with PBS. complete binding.

8. Add 50 μL of the antigen solution to the wells (the C. Add primary capture antibody (as above).
antigen solution should be titrated). All dilutions
should be done in the blocking buffer (3% BSA/PBS). 3. Wash the wells twice with PBS. A 500 mL squirt
Incubate for at least 2 hours at room temperature in bottle is convenient for this. The antibody solution
a humid atmosphere. washes can be removed by flicking the plate over a
9. Wash the plate four times with PBS. suitable container.
10. Add the labeled second antibody. The amount to be 4. The remaining sites for protein binding on the
added can be determined in preliminary experiments. microtiter plate must be saturated by incubating
For accurate quantitation, the second antibody with blocking buffer. Fill the wells to the top with 3%
should be used in excess. All dilutions should be done BSA/PBS with 0.02% sodium azide. Incubate for 2
in the blocking buffer. hours to overnight in a humid atmosphere at room
11. Incubate for 2 hours. or more at room temperature in temperature.
a humid atmosphere. 5. Wash wells twice with PBS.
12. Wash with several changes of PBS. 6. Add 50 μL of the standards or sample solution to the
13. Add substrate as indicated by manufacturer. wells. All dilutions should be done in the blocking
After suggested incubation time has elapsed, buffer (3% BSA/PBS with 0.05% Tween®-20).
chemiluminescence or optical densities at target Note: Sodium azide is an inhibitor or horseradish
wavelengths can be measured on an ELISA plate peroxidase. Do not include sodium azide in buffers or
reader. wash solutions, if an HRP-labeled conjugate will be
used for detection.
85
SAMPLE PROTOCOLS

7. Add 50 μL of the antigen-conjugate solution to 9. Add substrate as indicated by manufacturer.

the wells (the antigen solution should be titrated). After suggested incubation time has elapsed,
All dilutions should be done in the blocking buffer chemiluminescence or optical densities at target
(3% BSA/PBS with 0.05% Tween®-20). Incubate for wavelengths can be measured on an ELISA reader.
at least 2 hours at room temperature in a humid N
 ote: Competitive ELISAs yield an inverse curve,
atmosphere. where higher values of antigen in the samples or
8. Wash the plate four times with PBS. standards yield a lower amount of color change.

4.5 Multiplexed Bead-based 10. Incubate, then add 50 µL SA-PE

Detection 11. Wash 2-3 times and apply vacuum

A typical protocol can be found in MILLIPLEX® map 12. Add 100 μL of Sheath Fluid (or Drive Fluid if using
multiplex assay panels (EMD Millipore), which are MAGPIX® instrument) to all wells. Resuspend the
preconjugated, analytically validated panels with beads on a plate shaker for 5 minutes.
included quality controls, reagents, and all other 13. Acquire data on a Luminex™ instrument.
components to detect multiple analytes in a small
14. Save and analyze the Median Fluorescence Intensity
sample volume (5 - 50 µL) in a single kit, using a single
(MFI) data (Note: make sure all lysate samples in
catalogue number.
comparison are diluted by the same factor.)
1. Follow standard protocols for preparation of cell
and tissue lysates. It is recommended that protease
inhibitors (Cat. No. 535140, available separately) and
phosphatase inhibitors (Cat. No. 524629, available Tech Tips
separately) be added immediately prior to use. • When using stored samples, keep at -20°C
2. Dilute pre-cleared lysates at least 1:1 in Assay in “frosted” freezer and avoid multiple (>2)
Buffer. The suggested working range of protein freeze/thaw cycles.
concentration for the assay is 2.5 to 15 μg of total
protein/well (25 μL/well at 100 to 600 μg/mL). No • Thaw frozen samples completely, mix well by
additional dilution is necessary for the prepared vortexing and centrifuge before use.
Lysate Controls.
• Store samples in polypropylene tubes. DO NOT
3. Block the 96-well plate with 200 μL Assay Buffer. STORE SAMPLES IN GLASS TUBES.
Seal and place on a plate shaker to mix for 10
minutes at room temperature (20 - 25°C). • Add Protease Inhibitors to lysis buffer and
Na3VO4 in to inhibit Phosphatases
4. Apply vacuum/decant plate.
5. Add 25 µL standards, controls and samples to • For blood samples, allow the blood to clot
mapped out wells for at least 30 minutes before centrifugation.
6. Vortex bead bottle and add 25 μL of the Premixed Remove serum and assay immediately or
Beads to each well (Note: During addition of Beads, aliquot. Care must be taken when using
shake bead bottle intermittently to avoid any settling) heparin as an anticoagulant since an excess
(capture antibody) of heparin will provide false high values. Do
7. Incubate on a plate shaker for 2 hours at room not use more than 10 IU heparin per 1 mL of
temperature (20 - 25°C) or overnight at 4°C. blood collected.
8. Wash 2 - 3 times and apply vacuum or aspirate
9. Add 50 µL Detection Antibody cocktail

86
 SAMPLE PROTOCOLS
4.6 Immunohistochemistry/ 6. Prepared slides can be stored dry at -70°C until
stained. Equilibrate to room temperature and briefly
Immunocytochemistry re-dry prior to rehydration and staining.
Fresh Frozen (then fixed) Tissue Sections
1. Snap-freeze small tissue blocks (5x5x3 mm) in liquid
Paraffin-embedded Sections
nitrogen.
1. Conventional deparaffinization and dehydration
2. Transfer to cryostat and cut thin (5–30 μm) sections. sequence:
3. Collect specimens on clean poly-L-lysine-coated glass A. Incubate sections in xylene: 2 to 3 changes,
slides and dry at room temperature overnight (if you 5 min. each.
want to stain the same day let air-dry for 1–2 hrs.
B. 100% absolute ethanol: 2 changes, 3 min. each
until completely dry). Thorough drying is essential for
good adhesion to the slides. C. 95% ethanol: 2 changes, 3 min. each

4. Fix sections in acetone or absolute ethanol at 4°C for D. 80% ethanol: 3 min.
15 min. Use fresh ethanol or acetone for every 10–15 E. 50% ethanol: 3 min.
slides for best results. The organic solvents absorb F. Rinse with distilled water, PBS, or Tris buffer:
moisture from the air and tissue, as they do so, they 2 changes, 3 min. each.
lose their ability to fix the tissue effectively.
Note: Once sections have been rehydrated, do not

5. Thoroughly air-dry at room temperature or on mild allow them to dry.
heat (30–37°C). It is during this stage that much of
the chemical fixation is being finalized; improper
air-drying will lead to “soft” sections and likely loss of 2. Place slides in prewarmed (37°C) 0.1% trypsin in PBS
proper reactivity. for 5–60 min. or 0.4% pepsin in 0.01N HCl for 30 min.
6. Proceed with immunostaining or freeze. to one hour. Follow by rinsing with distilled water
3. If peroxidase conjugate is used, endogenous
Fixed, Frozen Tissue Sections peroxidase should be blocked at this stage.
1. Fix tissue either by perfusion with fixative or by Peroxidase activity results in the decomposition of
immersion in fixative for a set time period. Most hydrogen peroxide (H2O2). It is a common property
commonly, 4% Paraformaldehyde (PFA) solutions are of all hemoproteins such as hemoglobin, myoglobin,
used. cytochrome and catalases. Suppression of endogenous
peroxidase activity in formalin-fixed tissue entails
2. Fixed tissue is then prepared for cryoprotection by the incubation of sections in 3% H2O2 for 8–10 min.
submerging the target tissue in a hydrostabilizing Methanolic H2O2 treatment (1 part 3% H2O2 plus 4
solution. The cryoprotection is complete when the parts absolute methanol) for 20 min. can also used,
target tissue no longer floats in the stabilizing but it is not recommended for specimens where
solution. Because it works well and is relatively cell surface markers are to be stained. Methanolic
inexpensive, PBS+sucrose solutions ranging from treatment may also detach frozen sections from their
10% (less protection), to 30% (w/v) sucrose (greater carrier glass.
protection) are often used.
4. Wash twice with PBS.
3. Once stabilized, tissues can be removed from the
protectant solution and frozen at -70°C until 5. Proceed with immunostaining procedure.
sectioned.
4. Sectioned via cyrostat (5 – 40 μm*), where sections Staining with Polyclonal Rabbit or
can be collected directly onto slides, or floated onto Monoclonal Mouse Primary Antibody
slides via a PBS/waterbath. Usually up to 3 sections The following general protocol is intended for use as a
per slide can be placed; each spaced well apart. The guideline in developing antibody-specific procedures.
spacing prevents reagent mixing between samples. Different antibodies and tissues may require changes to
*Individual skill and tissue type will determine the this procedure. Review of individual product datasheets
thickness of the sections. Sections between and relevant literature references may be helpful in
10–15 μm provide the best results for clarity and customizing this procedure for specific applications.
integrity. Sections between 6–9 μm tend to tear during 1. Gently rinse slide containing sections with distilled
cutting, resulting in rough edges that can increase the water or buffer from a wash bottle. Place slide in room
background. Thicker sections while stronger during temperature buffer bath for 5 minutes to rehydrate
handling can be more difficult to stain. sections.
5. Sections on slides are thoroughly air-warmed/dried on 2. Using a Kimwipe®, gently remove excess liquid from
a slide warmer, usually overnight or at least 2 – 3 hrs. around the specimen. Avoid touching the tissue
at 40 – 50°C. directly.

87
SAMPLE PROTOCOLS

3. Apply 4 – 6 drops of normal serum, (normal serum 7. Rinse slide gently with distilled water or buffer from
from the host of the secondary antibody), diluted a wash bottle, and incubate in a buffer bath for 3 x 5
1:5-1:30 (final conc. 3% – 20%). Incubate for 20 – minutes (changing buffer in between washes).
30 minutes at 37°C. Note: For all procedures it is important to see that

4. Tap off serum and wipe away excess. Do not rinse. each step is adequately buffered, and that non-
5. Perform any antigen retrieval if necessary. reacted solutions are washed away after each step.

6. Apply 25 – 50 μL of rabbit (mouse) primary antibody, 8. Apply 25 – 50 μL of enzyme-conjugated antibody

diluted appropriately, per tissue section. Antibody directed against rabbit (mouse) immunoglobulins,
should cover sections completely. Incubate for diluted appropriately. Incubate 45 – 60 minutes.
desired time (see Antibody Staining in Section 3.7 for 9. Rinse slide gently with distilled water or buffer from
suggested parameters and temperatures). If optimal a wash bottle, and incubate in a buffer bath for 3 x
antibody dilution is unknown, perform a series of 5 minutes (changing buffer in between washes).
antibody dilutions in the range of 1:20 – 1:1,000 to 10. Apply substrate-chromogen solution and incubate
obtain initial results. until desired color intensity has developed.
Note: Antibody diluent is often very important for
 11. Rinse gently with distilled water from wash bottle.
consistent reactivity. Simple solutions are easier to Counterstain and coverslip.
troubleshoot then complex ones, thus antibodies
diluted only with simple buffers (PBS or TBS) are
usually recommended.

4.7 Flow Cytometry the conjugated antibody of interest. To the final

tube of cells, add the equivalent volume of 1x
Direct Staining Protocol for Flow Cytometry
PBS as conjugated antibody. This will serve as an
Note: The following protocol is given to provide a
autofluorescence control to establish the appropriate
general procedure that can be used as a template to
flow cytometer electronic settings. Vortex each of the
construct a specific protocol for an experimental assay.
sample tubes at moderate speed.
Investigators are strongly encouraged to collaborate with
a flow cytometry facility or technician to fully develop 3. Incubate samples for 20–30 minutes at room
appropriate procedures for their experimental systems. temperature or 4°C, in the dark.
4. Wash cells in tubes with 3 mL volumes of 1x PBS
with 1% BSA and centrifuge samples at 400 x g for
Items needed:
10 minutes at room temperature. Carefully pour
• C
 ells (~0.5–1 x 106/mL) prepared and counted, in PBS or pipette off supernatant fluid. This step can be
with Ca2+ and Mg2+, 1% BSA or 2% FCS. repeated if desired.
• D
 irectly conjugated monoclonal antibody to desired 5. Cells can be analyzed without fixing if they are to be
cell surface marker. used immediately. Simply resuspend the cell pellet
• W
 ash solution (1x Dulbecco’s PBS, Ca2+ and Mg2+ free in 500 μL of PBS wash solution, and keep at 4°C
with and without 1% BSA) in the dark until used (usually less than 4 hours).
Alternatively, loosen the cell pellet by vortexing
• O
 ptional fixative: 1% formaldehyde-PBS pH 7.4,
it in the residual wash fluid. Add 500 μL of 1%
freshly prepared.
formaldehyde-PBS to each sample and quickly mix
again. It is critical to mix the cell pellet prior to
Method: adding the fixative; otherwise the cells will become
1. Add 50-100 μL of cells to each of three 12 x 75 mm fixed into a solid mass that cannot be sent through
polypropylene or polystyrene tubes the flow cytometer. Fixed samples can be stored
at 4°C in the dark until flow cytometry analysis is
Note: The initial amount of cells collected should be
 performed. Fixed samples should be used within
adjusted to approximately ~0.75 x106 cells per mL so 48 hours.
that 50 μL added will equal approximately 105 cells
per tube.
2. To the first tube, add the appropriate volume (usually
5–10 μL) of fluorochrome-conjugated monoclonal
antibody. To the second tube, add the same volume
of matched isotype control antibody. The isotype
control antibody should match the isotype of

88
 SAMPLE PROTOCOLS
4.8 Functional Blocking and B. Antibody Neutralization
1. Prepare a 1:2 (16 points) or 1:4 (8 points) serial
Stimulation Assays dilution of IL-18 antibody in complete medium
Due to the immense complexity of protein structure
(starting at 300 nM) in a 96-well dilution plate.
function relationships, it is very hard to generalize a basic
protocol for functional antibody assays. 2. To a 96-well flat-bottom tissue culture plate, add
50 mL/well diluted IL-18 antibody in quadruplicate.
For lysate-based or recombinant protein based Include eight control wells containing 50 mL/well
neutralization studies, a rough guide would be to use complete medium without IL-18 antibody (IL-18-only
the neutralizing antibody at 103 times the concentration control wells) and eight control wells containing
of the target protein to achieve ND50 (50% neutralizing 100 mL/well complete medium without IL-18 antibody
dose). Serial dilutions of the antibody are necessary to (medium-control wells).
determine the ND50 curve. 3. Add 50 mL/well human IL-18 (8 ng/mL), except
to medium-control wells. Preincubate the plate
Typically, the antibody and antigen are combined first in containing IL-18 and IL-18 antibody for 1 h at 37°C,
suspension. Then serial dilutions of antibody are applied 5% CO2.
to a cell line or enzymatic substrate in culture to measure 4. Harvest KG-1 cells, centrifuge at 1,500 rpm for 5 min,
the resulting effect of the dose. aspirate off media, and resuspend at a cell density of
3 x 106 cells/mL in complete medium containing
General antibody neutralization protocol 20 ng/mL human TNF.
for determining compound bioactivity
This characteristic neutralizing antibody protocol used for 5. Add 100 mL KG-1 cells to each well in the 96-well
cytokine bioassays is taken from G.M. Veldman et al. in plate and incubate for 24 h at 37°C, 5% CO2.
R. Kontermann and S. Dübel (eds.), Antibody Engineering 6. Continue with section C.
Vol. 1.* Please consult that volume for more extensive
discussion on different function blocking protocols and C. Determination of IL-18 Bioactivity and
design considerations. It offers an excellent discussion and Antibody Neutralization
guide to function blocking antibodies and much more. 1. Transfer plate contents to 96-well V-bottom tissue
culture plates and centrifuge plates at 1,500 rpm for
Typical Bioassay to Determine Antibody 10 min. Transfer 150 mL of the culture supernatants
Neutralization Potency to 96-well flat-bottom tissue culture plates. Do not
(KG-1 Assay for IL-18) (Konishi et al. 1997; Fernandez-Botran and Vĕtvička 2001) disturb the cell pellets. At this point, the supernatants
The first step in setting up a bioassay is to determine can be stored at -20°C.
the biologic response of the KG-1 cell line to different 2. Determine human IFNg concentrations in the
concentrations of human IL-18 by measuring the then supernatants by using a human IFNg ELISA kit. Further
selected as the concentration to be used in the second dilution of supernatants may be necessary to ensure
part of the assay, in which anti-human IL-18 antibody is that data points fall within the standard curve of the
prepared at different concentrations. The potency of the ELISA.
neutralizing antibody is expressed as the IC50, which is
3. Calculate the EC50 or EC70 for IL-18 by plotting the
the antibody concentration that results in 50% inhibition
IL-18 concentration on the x-axis versus human IFNg
of the IL-18-induced IFNg response.
on the y-axis.
A. Determine the Biologic Response Curve for IL-18 4. Calculate the IC50 of the IL-18 antibody by plotting the
1. Prepare a standard curve for IL-18. In a 96-well antibody concentration on the x-axis versus human
dilution plate, prepare a serial 1:2 (16 points) or 1:4 IFNg on the y-axis. The maximum IFNg response can
(8 points) dilution of human IL-18 (starting at be determined from the IL-18-only control wells and
2,000 ng/mL) in complete medium. the background IFNg response can be determined from
the medium-control wells. The IC50 of the antibody
2. In a 96-well flat-bottom tissue culture plate, add
is the concentration that causes 50% inhibition of
100 mL/well human IL-18 dilutions in quadruplicate.
the maximum IFNg response over the background
Include eight control wells containing 100 mL/well
response.
complete medium without IL-18.
3. Harvest KG-1 cells, centrifuge at 1,500 rpm for 5 min,
aspirate off media, and resuspend at a cell density of
3 x 106 cells/mL in complete medium containing
20 ng/mL human TNF.
4. Add 100 mL KG-1 cells to each well in the 96-well
plate and incubate for 24 h at 37°C, 5% CO2.
5. Continue with section C.

89
Notes

90
 APPENDIX
Appendix Protein A/G Binding Affinities
Species Immunoglobulin Protein A Protein G
Bovine Ig ++ ++++
Chicken Ig - +
Goat Ig +/- ++
Making Serial Antibody Dilutions Guinea Pig Ig ++++ ++
Reagents/Equipment: Hamster Ig + ++
• PBS or other appropriate buffer. Mouse IgG1 + ++
• Small capped tubes IgG2a ++++ ++++
• Pipets capable of accurate delivery of 200 μL and IgG2b +++ +++
1000 μL volumes IgG3 ++ +++
IgGM +/- -
Keep buffer and tubes in ice Pig Ig +++ +++
1. Pipet 450 μL buffer into a tube, labeled as tube X. Rabbit Ig ++++ +++
2. Add 50 μL antibody solution, and mix. This gives a 1:10 Rat IgG1 - +
IgG2a - ++++
dilution of the antibody in tube X.
IgG2b - ++
3. Label tubes A through M for 1:50, 1:100, 1:200, 1:400,
IgG2c + ++
etc. to 1:204,800 dilutions.
IgGM +/- -
4. Pipet 1600 μL of dilution buffer into tube A (to
Sheep Ig +/- ++
become a 1:50 dilution). Pipette 1000 μL (1.0 mL) of
dilution buffer into tubes B through M (to become Key: - (none), +/- (very low), + (low) ---> ++++ (high)
1:100–1:102,400 dilutions).
5. Pipette 400 μL of 1:10 antibody dilution from tube X Useful Formulations for Western
into tube A (which contains 1600 μL buffer). Mix well.
This results in a 1:50 antibody dilution.
Blotting and Immunoprecipitation
Cell Lysis Buffer
6. Take 1000 μL of antibody sample from Tube A and add
50 mM Tris-HCl, pH8.0
to Tube B (which contains 1000 μL buffer). Mix well.
150 mM NaCl
7. Take 1000 μL of antibody sample from Tube B and add
1% NP-40 or Triton® X-100
to Tube C (which contains 1000 μL buffer), etc.
Mix well.
2x Immunoprecipitation Buffer
2% Triton® X-100
Sample to Volume of Volume of Resulting
Tube be diluted Sample Buffer Dilution 300 mM NaCl
A 1:10 400 μL 1600 μL 1:50 20 mM Tris, pH 7.4
B 1:50 1000 μL 1000 μL 1:100 1.0% NP-40
C 1:100 1000 μL 1000 μL 1:200 2 mM EDTA
D 1:200 1000 μL 1000 μL 1:400 2 mM EGTA
E 1:400 1000 μL 1000 μL 1:800 0.4 mM sodium vanadate (phosphatase inhibitor)
F 1:800 1000 μL 1000 μL 1:1,600 0.4 mM PMSF
G 1:1,600 1000 μL 1000 μL 1:3,200
H 1:3,200 1000 μL 1000 μL 1:6,400 2x SDS-PAGE Sample Buffer
I 1:6,400 1000 μL 1000 μL 1:12,800 100 mM Tris-HCl, pH 6.8
J 1:12,800 1000 μL 1000 μL 1:25,600 2% Sodium Dodecyl Phosphate (SDS)
K 1:25,600 1000 μL 1000 μL 1:51,200
20% Glycerol
L 1:51,200 1000 μL 1000 μL 1:102,400
0.2% Bromophenol Blue
M 1:102,400 1000 μL 1000 μL 1:204,800
2–10% b-mercaptoethanol (or DTT)

100x Protease Inhibitor Cocktail

PMSF 5 mg (50 μg/mL)
Aprotinin 100 μg (1.0 μg/mL)
Leupeptin 100 μg (1.0 μg/mL)
Pepstatin 100 μg (1.0 μg/mL)
Add 100% Ethanol to 1 mL. Aliquot and store at -20°C.

Recommended Acrylamide Gel Percentages

% Acrylamide Protein Size Range

8% 40–200 kDa
10% 21–100 kDa
12% 10–40 kDa

91
APPENDIX

Recipes for Common Fixatives Carnoy’s Fixative

Caution: Formaldehyde is toxic and should be handled 1. 10 mL of glacial acetic acid
with caution under a chemical fume hood. Consult 2. 30 mL of chloroform
Material Safety Data Sheets for proper handling of all 3. 60 mL of absolute alcohol (100% Ethanol)
laboratory chemicals. 4. Mix

4% Paraformaldehyde (PFA) PLP (Periodate-lysine-paraformaldehyde) Fixative

1. Heat 250 mL of double strength phosphate buffer 1. Dissolve 7.3 g of lysine monohydrochloride in 200 mL
stock solution (see step 4) to 140°F (60°C) in a beaker of ddH2O.
with a disposable stir bar in a hood. 2. Adjust pH to 7.4 with 0.1 M Na2HPO4 (Na2HPO4·2H2O
2. Add 20 g granular paraformaldehyde and stir until it is 17.8 g/L) NOT phosphate buffer!
dissolved. 3. Complete volume to 400 mL with 0.1 M phosphate
3. Add 250 mL deionized water and filter the solution buffer pH 7.4. This lysine-phosphate buffer keeps
into a container placed on ice. The solution is ready for 2–3 days in the refrigerator, but can be frozen in
when cold. Adjust pH to 7.0–7.4. aliquots for longer storage.
4. Double Strength Phosphate Buffer Stock solution 4. Just before use, mix 375 mL lysine-phosphate buffer
isprepared by dissolving 7.7 g NaOH and 33.6 g with 100 mL 20% Formaldehyde and top to 500 mL
NaH2PO4·2H2O in 1 liter deionized water. with ddH2O. Add 1.06 g sodium periodate (NaIO4)
and mix well. The PLP fixative must be used within
4% Paraformaldehyde with 2% Glutaraldehyde maximum 2 hrs.
1. Heat 250 mL double strength phosphate buffer stock Final concentrations: Lysine 75 mM, formaldehyde 4%,
solution (see above) to 140°F (60°C) in a beaker with a sodium periodate 10 mM.
disposable stir bar. (Note: Some PLP formulations in literature also use
2. Add 20 g granular paraformaldehyde and 10 g 2% paraformaldehyde)
glutaraldehyde and stir until it is dissolved.
3. Add 250 mL deionized water and filter the solution Acetone/Methanol Fixative
into a container placed on ice. The solution is ready 1. 100 mL acetone
when cold. Adjust pH to 7.0–7.4. 2. Add 100 mL methanol
3. Mix well. Use fresh. 50–50 solution is used at room
Buffered Formaldehyde (Formalin) temperature or -20°C
1. Dissolve 32.5 g Na2HPO4·2H2O and 20 g NaH2PO4·2H2O
in 4.5 L deionized water.
2. Add 500 mL 40% Formaldehyde.
3. Mix; Adjust pH to 7.0–7.4.

Bouin’s Fluid
1. Picric Acid (standard aqueous solution) 75 mL.
2. Formalin (40% aqueous Formaldehyde) 20 mL
3. Glacial Acetic Acid 5 mL.
4. Mix

92
 APPENDIX
Glossary Cross-reactivity – The binding of an antibody Immunoprecipitation – A technique that uses
or population of antibodies to epitopes on other antibodies to bind antigens in solution to yield a
antigens. precipitating complex, the components of which can
Adjuvant – Any substance that has the capacity to then be isolated.
Denatured – The conformational change in an
enhance the immune response to an antigen.
antigen that may expose or destroy an epitope. Monoclonal antibody – A homogeneous population
Affinity – The strength of reaction between antibody of antibodies that are raised by the fusion of B cells
Detection antibody – A primary antibody used with immortal cell cultures to produce hybridomas.
and antigen at a single antigenic site.
in ELISA procedures to allow secondary antibody
Affinity purification – Column purification where labeling of antigen. Negative control sample - any tissue, cell line,
specific antibody fraction binds to the antigen to lysate or purified protein that is known from previous
ELISA (Enzyme-Linked Immunosorbent Assay) work to be void of the antigen of interest.
which it was made.
– A serological technique that uses antibodies or
Affinity constant – Describes the binding antigens to capture and quantify the amount of Neutralization – The mechanism of antibody
interaction between antibody and antigen. Also antigen or antibody in unknown samples. binding to sites on pathogen to prevent its growth
known as association or equilibrium constant. and/or its entry into cell. Normal serum – Blood
Epitope – The small site on an antigen where a serum extracted from non-immunized animals; often
Allergen – An antigen that elicits hypersensitivity or complementary antibody binds via its variable used as a control.
allergic reaction. region.
Oposonization – The coating of pathogen surface
Antibody – An immunoglobulin capable of specific Flow cytometry – A technique that often uses with any molecule, which can make it more readily
combination with the antigen that caused its fluorescent antibodies to label whole cells in digestable by phagocytes.
production in a susceptible animal. suspension for measurement and sorting as they
pass by a tuned light source. Optimal working dilution – Concentration (dilution)
Antigen – Any substance foreign to the body that that maximizes the positive signal while minimizing
elicits a specific immune response. Fluorochrome (fluorophore) – Molecular label on background (negative) reactivity.
antibody that emits a distinct, measurable color
Antigen–Antibody complex – A non-covalent spectrum in response to a specific laser or chemical Peptide – A small amino acid sequence used for
association of antibody and antigen molecules (also interaction. generating sequence-specific antibodies.
known as immune complex).
Hapten – Small molecule incapable of eliciting a Phagocytes – Specilized cells that perform
Antigen retrieval – Procedure designed to enhance specific antibody response without being chemically phagocytosis, e.g., neutrophils and macrophages.
the binding of antibody to antigen in tissue typically coupled to a larger carrier protein.
in response to an epitope-blocking fixation or Plasma Cells – Terminally differentiated B cells that
embedment technique. Hinge region – Flexible domain of antibody that produce antibodies.
binds Fab arm to Fc region.
Antiserum – Blood from an immunized host Polyclonal antibody – Multiple B cell response to an
presumably possessing antibodies of interest as well Hybridoma – Hybrid cell lines that make specified antigen resulting in a mixture of antibodies typically
as other serum proteins. monoclonal antibodies. They are formed by fusing recognizing a variety of epitopes on the antigen.
a specific antibody-producing B lymphocyte with a
Ascites fluid – Unpurified monoclonal myeloma cell that grows in tissue culture without Positive control sample – Any tissue, cell line, lysate
antibodycontaining fluid drawn directly from making its own immunoglobulin chains. or purified protein that is known from previous work
hybridomas grown within a living host. to contain and bind the antigen of interest in a
Hybridoma supernatant – Monoclonal antibody- particular application.
Avidity – A measure of the overall strength of containing fluid collected from hybridoma cell
binding of the entire antibody-antigen complex. cultures. Pre-immune serum – Blood serum extracted prior
to that animal’s immunization with an antigenic
B Cells – B lymphocytes that differentiate into Immunity – Ability to resist infection to a particular substance; often used as a control.
antibody producing cells upon activation by antigen. antigen.
Primary antibody – The antibody that directly binds
Capture antibody – An anchored primary antibody Immunoblotting – A technique that uses antibodies the antigen of interest.
used in ELISA procedures to bind an antigen in to probe proteins transferred from an electrophoresis
solution. gel onto membrane. For example: Western blotting. Protein A – A cell membrane component of
Staphylococcus aureus that binds to the FC region
Carrier protein – Typically a large highly antigenic Immunocytochemistry – A technique that uses of IgG
molecule used in combination with a small antigen antibodies to probe specific antigens in live or fixed
or hapten to cause a larger, faster specific immune cell cultures. Protein A/G purification – Column purification
response in an immunized animal. where the Fc domain of antibodies binds to the high
Immunodiffusion – A technique to detect antigen affinity S. aureus protein A or G.
Chemiluminescent – Producing light through or antibody by formation of an antigen-antibody
a chemical reaction catalyzed by an enzyme; precipitate in an agar gel. Secondary antibody – Typically the labeled antibody
chemiluminescent substrates require X-ray film or that binds to the antigen-binding primary antibody.
other light capturing devices for detection. Immunofluorescence – A technique for detection of A secondary may also bind to streptavidin, which is
antigen molecules using antibodies labeled with a bound to the primary.
Chromogen – The chemical substrate that changes fluorescent dye.
color in the presence of a specific enzyme-tagged Specificity – The likelihood that the particular
antibody. For example: DAB. Immunogenic – A description of an antigen’s ability antibody is binding to a precise antigen epitope.
to stimulate antibody production.
Complement – A set of plasma proteins that act in T Cells – T lymphocytes that originate in thymus.
a cascade of reactions to attack extracellular forms Immunoglobulin (Ig) – General term for a number
of classes of proteins that functions as antibodies. Valency – A description of the relative ability of an
of pathogens.
antibody to interact with antigens, usually related to
Conformational Epitope – Epitopes on an antigen Immunohistochemistry – A technique that uses the number of available variable regions.
formed from several separate regions in the primary antibodies to probe specific antigens in fresh frozen
or processed tissue. Western blot – Detection, using a specific antibody,
sequence of a protein brought together by protein
of proteins separated by electrophoresis and then
folding.
bound to a membrane.

93
Notes

94
Notes

95
Notes

96
Reput(Ab)le
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Lit. No. PB5736EN00 BS-GEN-13-08821 11/2013 Printed in the USA.
© 2013 EMD Millipore Corporation, Billerica MA USA. All rights reserved. V 1.0