Analytical Microbiology
MIC 402
K.M. Mazharul Alam
Lecturer,
MNS Department,
BRAC University
Protein Characterization
K.M. Mazharul Alam
Lecturer,
MNS Department,
BRAC University
2
Iso-electric Focusing (IEF)
MW Versus Charge
Enolase (NP 417259 )
# Phosphates Molecular Weight Isoelectric Point
0 45650.4172 5.32
1 45728.3812 5.25
2 45806.3452 5.19
4
Isoelectric Points Of Some Proteins
Pepsin 1.0
Egg albumin 4.6
Serum albumin 4.9
Urease 5.0
B-Lactoglobulin 5.2
Hemoglobin 6.8
Myoglobin 7.0
Chymotrypsinogen 9.5
Cytochrome C 10.7
Lysozyme 11.0
Credit:: Lehninger PoB, 3rd Edn
p.135
Iso-electric Focusing (IEF)
◼ High resolution technique for separating proteins.
◼ Combines Isoelectric point and electric field.
◼ Proteins are subjected to electric field in a pH gradient.
Iso-Electric Point
◼ The pH at which net charge on protein becomes zero.
◼ Below pI – Positive charge.
◼ Above pI – Negative charge.
◼ Proteins move toward the electrode with the opposite
charge.
Iso-Electric Focusing (IEF)
◼ All proteins have an isoelectric point pH.
◼ A procedure to determine the isoelectric point of proteins thus, a
mixture of proteins can be electrophoresed through a solution having a
pH gradient from the anode to the cathode and each protein will migrate
to the position in the pH gradient according to its isoelectric point. This
is called Isoelectric focusing.
◼ Protein migrate into the point where its net charge is zero-isoelectric pH.
ISO-ELECTRIC FOCUSING (IEF)
Credit: Lehninger PoB, 3rd Edn p.135
2D Gel Electrophoresis
◼ Most sophisticated analytical method for separating proteins
◼ Combines the techniques of IEF and SDS-PAGE
◼ In the 1st dimension is IEF, carried out in polyacrylamide gels in narrow
tubes
◼ Gel is extruded from the tube and incubated in a buffer containing SDS
◼ Placed top of SDS-polyacrylamide gel along the stacking gel (2nd
dimension)
Credit: Lehninger PoB, 3rd Edn p.135
2D Gel Electrophoresis : Significance
◼ Provide map of intact proteins which reflects changes in protein
expression, isoforms or post translational modification.
◼ Resolve more than 5000 protein spots simultaneously (~2000 routinely)
◼ Able to resolve proteins with pI around 0.001 pH units
◼ Detect and quantify <1ng of protein per spot
◼ Good for identifying novel proteins
◼ This technique has led to the introduction of the term Proteome which
describes the complete set of proteins expressed by the genome (full
nucleic acid component of a species) in an organism at a given time.
2d Gel Electrophoresis: Limitations
◼ Cannot handle extremely acidic/ basic proteins
◼ Misses some large proteins & membrane proteins
Blotting
◼ Blots are techniques for transferring DNA , RNA and proteins onto a
carrier so they can be separated, and often follows the use of a gel
electrophoresis.
◼ There are four major types of blotting
1. Western blotting
2. Southern blotting
3. Northern blotting
4. Eastern blotting
Northern blotting:
to detect specific RNA molecules
among a mixture of RNA
Western blotting:
Eastern blotting:
to detect specific proteins in a
to analyze protein post-
sample of tissue homogenate or
translational modifications (PTM)
extract
Southern blotting:
to detect a specific DNA molecules
in a blood or tissue sample.
Western Blotting
◼ PAGE does of course achieve fractionation of a
protein mixture during the electrophoresis
process.
◼ It is possible to make use of this fractionation
to examine further individual separated
proteins.
◼ The first step is to transfer or blot the pattern
of separated proteins from the gel onto a sheet
of nitrocellulose paper. The method is known as
protein blotting or western blotting.
◼ Transfer of the proteins from the gel to
nitrocellulose is achieved by a technique known
as electroblotting.
Western Blotting
Cell Lysis by Detergents
Cell Removal and Sonication
◼ Steps in western blotting:
Cells in Culture Human Cells Containing
1. Tissue preparation Heat
Protein
SDS -
- -
2. Gel electrophoresis
Denaturation of
Proteins
- - - -
- - - - - -
3. Transfer - -
Detergents Bind
4. Blocking Proteins
5. Detection
6. Analysis
Load Proteins on
Gel
Apply Electric Current
- ---
--
+ Proteins Separate by Size
Proteins Separate by Size
01.Tissue Preparation
◼ Samples may be taken from whole tissue or from cell culture.
◼ In most cases, solid tissues are first broken down mechanically using a
blender. However, it should be noted that bacteria, virus or environmental
samples can be the source of protein and thus Western blotting is not
restricted to cellular studies only.
◼ Assorted detergents, salts, and buffers may be employed to encourage lysis
of cells and to solubilize proteins.
◼ Tissue preparation is often done at cold temperatures to avoid protein
denaturing.
02. Gel Electrophoresis
◼ The SDS PAGE technique is a prerequisite for Western
blotting.
◼ The proteins of the sample are separated using gel
electrophoresis. Separation of proteins may be by
isoelectric point, molecular weight, electric charge, or a
combination of these factors.
Cell Lysis by
Detergents and
Cell Removal Sonication
Cells in Human Cells Containing
Culture Protein
SDS -
Heat or - -
Denaturation of - LDS - -
- - - Proteins - -
- -
- -
Detergents Bind
Proteins
Load Proteins on
Gel
Apply Electric
- ---
Current
--
+ Proteins Separate by Size
03.Transfer: Membrane
◼ Membranes used for transfer
1. Nitrocellulose
2. PVDF (polyvinylidene difluoride)
▪ Both varieties of membrane are chosen for their non-specific protein binding
properties (i.e. binds all proteins equally well). Protein binding is based upon
hydrophobic interactions, as well as charged interactions between the
membrane and protein.
▪ Nitrocellulose membranes are cheaper than PVDF , but are far more fragile
and do not stand up well to repeated probings.
03.Transfer:Transfer Methods
Transfer of proteins is done by electroblotting
Three methods of transfer :
▪ Wet or Tank Transfer
▪ Semi dry transfer
▪ Dry Transfer
More about transfer methods: https://www.thermofisher.com/bd/en/home/life-science/protein-biology/protein-biology-
learning-center/protein-biology-resource-library/pierce-protein-methods/western-blot-transfer-methods.html
03. Transfer: Membranes
Criteria Nitrocellulose PVDF
Binding Capacity 80 to 100 μg/cm2 170 to 200 μg/cm2
Sensitivity Lower Higher
Background noise Lower Higher
hydrophobic interactions hydrophobic and dipole
Binding interactions
( requires methanol) interactions
more durable and has higher
Physical characteristics brittle and fragile
chemical resistance
Typical (0.1, 0.2 or 0.45μm)
Typical (0.1, 0.2 or 0.45μm)
Pore size (ideal for high MW proteins)
(ideal for low MW proteins)
Transfer: System
◼ In this method a sandwich of gel and
nitrocellulose is compressed in a cassette and
immersed, in buffer, between two parallel
electrodes.
◼ A current is passed at right angles to the gel,
which causes the separated proteins to
electrophorese out of the gel and into the
nitrocellulose sheet.
◼ The nitrocellulose with its transferred
protein is referred to as a blot. Once
transferred onto nitrocellulose, the
separated proteins can be examined further.
Credits: Wilson & Walker, PToBMB, 6th ed, pg. 470
04.Blocking
◼ The membrane has the ability to bind to different types of
proteins. So blocking is performed so avoid unwanted binding.
◼ The blot is first incubated in a protein solution, for example 10%
(w/v) bovine serum albumin, or 5% (w/v) non-fat dried milk, which
will block all remaining hydrophobic binding sites on the
nitrocellulose sheet.
05.Detection
◼ Detection is done in two steps:
(1) Primary antibody
The blot is incubated in a dilution of an antiserum (primary antibody) directed against
the protein of interest. This IgG molecule will bind to the blot if it detects its
antigen, thus identifying the protein of interest.
(2) Secondary antibody
In order to visualize this interaction the blot is incubated further in a solution of a
secondary antibody, which is directed against the IgG of the species that provided
the primary antibody.
05.Detection
◼ For example, if the primary antibody was raised in a rabbit
then the secondary antibody would be anti-rabbit IgG. This
secondary antibody is appropriately labelled so that the
interaction of the secondary antibody with the primary
antibody can be visualized on the blot.
06. Analysis
◼ One of the most common detection methods is to use an enzyme-linked
secondary antibody.
◼ In this case, following treatment with enzyme-labelled secondary antibody, the
blot is incubated in enzyme–substrate solution, when the enzyme converts the
substrate into an insoluble colored product that is precipitated onto the
nitrocellulose.
◼ The presence of a colored band therefore indicates the position of the protein
of interest.
06. Analysis
◼ The enzyme used in enzyme-linked antibodies is usually either alkaline
phosphatase or horseradish peroxidase
Alkaline phosphatase
◼ which converts colorless 5-bromo-4-chloro-indolylphosphate (BCIP) substrate
into a blue product
Horseradish peroxidase
(i) Colorimetric assay: with H2O2 as a substrate, oxidizes either 3-amino-9-
ethylcarbazole into an insoluble brown product, or 4-chloro-1-naphthol into an
insoluble blue product.
Credits: Wilson & Walker, PToBMB, 6th ed, pg. 471
06.Analysis
(ii) Chemiluminescence assay: An
alternative approach to the detection of
horseradish peroxidase is to use the
method of enhanced chemiluminescence
(ECL).
◼ In the presence of hydrogen peroxide
and the chemiluminescent substrate
luminol, horseradish peroxidase oxidizes
the luminol with concomitant production
of light, the intensity of which is
increased 1000-fold by the presence of
a chemical enhancer.
◼ The light emission can be detected by
exposing the blot to a photographic film.
Credits: Wilson & Walker, PToBMB, 6th ed, pg. 472
- -- - --
Transfer or Blot Protein
-- --
from Gel to
Nitrocellulose and/or
PVDF Membrane
Block
Membrane with
Non-Specific
Proteins
Incubate Membrane
- -- - --
with 1o Antibody
-- --
1o Antibody is a Rabbit
Anti-Human b-Actin
1o Antibody Binds Antigen Antibody Non-Specific Proteins Bind
(i.e. Protein of Interest) to Unbound Regions of
Membrane
- -- - --
Add HRP-Conjugated 2o
Antibody
-- --
2o Antibody is a Goat
Anti-Rabbit-HRP-
Conjugated Antibody
Add
Chemiluminescent
Substrate Luminol Light
Detected by
Film
HRP
- -
Applications
◼ The confirmatory HIV test employs a western blot to detect anti-HIV antibody
in a human serum sample. Proteins from known HIV-infected cells are separated
and blotted on a membrane. Then, the serum to be tested is applied in the
primary antibody incubation step; free antibody is washed away, and a secondary
anti-human antibody linked to an enzyme signal is added. The stained bands then
indicate the proteins to which the patient's serum contains antibody.
◼ Western blot can also be used as a confirmatory test for Hepatitis B infection.
Limitations
◼ If a protein is degraded quickly, western blotting won't detect it well.
◼ This test takes longer than other existing tests It might also be more
costly.
◼ Many steps where errors can occur.
◼ Large amount of sample needed (5-50 mg)
Protein Characterization
Southern Blotting
Objective
◼ Transferring the DNA from the intact gel onto a piece of nitrocellulose
or nylon membrane placed in contact with it.
◼ This transfer, named a Southern blot after its inventor Ed Southern
Why?
◼ This provides a more permanent record of the sample, since DNA begins
to diffuse out of a gel that is left for a few hours.
Southern Blotting
◼ DNA is applied to an agarose gel, and electrophoresis separates the fragments
of DNA according to size.
◼ The gel is then placed atop a thin sponge wick resting in a dish of salt solution, and a
special filter (typically nitrocellulose) is placed on top of the gel.
◼ A stack of absorbent material (typically paper towels) is placed on top of this stack.
The absorbent material draws the salt solution from the dish into the wick and through
the gel by capillary action, which transfers the DNA fragments into the filter. The
procedure is called a "Southern transfer" after the scientist
Eric Southern who invented the procedure. The filter now contains
the DNA fragments in the same pattern as the gel, but is more easily manipulated
Procedure: Step By Step
Step-1
◼ The gel is soaked in alkali to render the DNA single stranded. (Alkali denatures DNA)
Step-2
◼ It is then transferred to the membrane so that the DNA becomes bound to it in exactly
the same pattern as that originally on the gel.
◼ The DNA being single stranded (SS) is now ready for hybridization.
Step-3
◼ The membrane can now be treated with a labelled DNA molecule, for example a gene
probe.
◼ The probe also has to be SS
Procedure: Step By Step
Step-4
◼ This single-stranded DNA probe will hybridize under the right conditions to
complementary fragments immobilized onto the membrane.
Step-5
◼ A series of washing steps with buffer is then carried out to remove any
unbound probe.
Step-6
◼ Membrane is developed, like a photograph, after which the precise location
of the probe and its target may be visualized
Nucleic Acid Characterization
Northern Blotting
Objective
◼ Identification of specific mRNA sequences of a defined length
by hybridization to a labelled gene probe
◼ It is possible with this technique not only to detect specific
mRNA molecules but also to quantify the relative amounts of
the specific mRNA.
Procedure
◼ separate the mRNA transcripts by gel electrophoresis under denaturing
conditions
◼ transfer from gel to direct slots on a specific blotting apparatus containing
the nylon membrane
◼ This is termed slot or dot blotting and provides a convenient means of
measuring the abundance of specific mRNA transcripts
◼ however, it does not provide information regarding the size of the
fragments
Your learnings
◼ Isotachopohoresis
◼ Iso electric focusing (IEF) ▪ Principle
◼ 2D gel electrophoresis ▪ Procedure
◼ Blotting techniques
1. Western blotting
▪ Objectives
2. Southern blotting ▪ Principle
3. Northern blotting ▪ Procedure
