BIO345

Cell & Molecular Biology Lab

LAB 7&8
Western Blot

Prepared by Dr. Rayan M. Naser & Dr. Oula El Atat

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Introduction
What is Western Blotting?

A technique in which proteins are separated by gel

electrophoresis and transferred to a membrane sheet. A specific
protein is then identified through its reaction with labeled
antibody.

Verify Protein Determine Protein Analyze Protein:

Expression concentration Protein Interaction

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 Analyzing protein-protein interaction using far-WB, extra
A labeled or antibody-detectable "bait" protein is used to probe and detect a target "prey" protein on the membrane(your
protein of interest).
Proteins in a cell lysate containing prey proteins are firstly separated by SDS or native PAGE, and transferred to a membrane,
as in a standard WB. The proteins in the membrane are then denatured and renatured. The membrane is then blocked and
probed, usually with purified bait protein(s).
The bait proteins are detected on spots in the membrane where a prey protein is located if the bait proteins and the prey
protein together form a complex.
 

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Introduction
Components of the System

Glass Plates, Spacers, and Combs

Reservoir/Tank
Molecules to be separated
–Proteins
Support medium
–Gel (Polyacrylamide)
Buffer System Power Supply
–High Buffer Capacity

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 Steps in a Western
Blotting
Western blotting consists of a series of incubations with different
immunochemical reagents separated by wash steps

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Detection of specific proteins: SDS-PAGE and Western blot

1. Separate proteins by SDS PAGE

2. Transfer proteins to membranes (i.e. Nitrocellulose and/or PVDF )
3. Block non-specific sites on membrane
4. Incubate with primary antibody, wash
5. Incubate with secondary antibody, wash
6. Detect secondary antibody

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SDS-PAGE and Membrane Transfer
-
Cell Removal Cell Lysis SDS -
-
-
- -
Cells in Culture Human Cells Containing Protein -
-
Detergents Bind
Proteins
- --
Transfer Protein from Heat
Gel to Membrane Denaturation of
- - -- Proteins
-
- - -
--

-
- -

Load Proteins on Gel

---
Set transfer sandwich
Electric
--
Current
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What is Membrane Blotting (Transfer)?

Two major factors affect the efficiency:

1. The elution from the gel
• Use the lowest percentage of acrylamide that will allow resolution
• high molecular weight proteins blot poorly

2. Efficiency of binding to the membrane

• Nitrocellulose
• Polyvinylidene fluoride (PVDF)

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Membranes used for Blotting (Transfer)

• Nitrocellulose:
• One of the first membrane types used for western blotting

• PVDF membranes:
• Developed in 1985
• Improved protein retention under harsh conditions (organic solvents, acidic or basic conditions).
• Greater mechanical strength
• Chemical stability
• Offers advantages when stripping and reprobing.

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 Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

Incubate Membrane
- -- Block Membrane with --- with 1 Antibody ---
Non-Specific Proteins

-- -- 1o Antibody is a --
Rabbit Anti b-
Actin Antibody
Non-Specific Proteins Bind to
Chemiluminescent Unbound Regions of Membrane
Substrate is added
Add HRP-Conjugated
2 Antibody
---

2o Antibody is a Goat
- - Anti-Rabbit-HRP-
Conjugated Antibody
1o Antibody Binds Antigen
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(i.e. Protein of Interest)
Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

• After transfer, the remaining surface of the membrane is blocked

•  prevent nonspecific binding of antibodies

• A variety of blocking buffers:

 5% BSA
 Because of its low cost, 5% milk is a popular blocking buffer

• The blocking buffer should improve the sensitivity of the assay by reducing background interference and
improving the signal to noise ratio.

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Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

Western blotting is typically performed by probing the blocked membrane with a primary antibody that
recognizes a specific protein or epitope on a group of proteins

Structure:
2 heavy chains + 2 light chains Disulfide bonds
2 antigen binding sites
Immunoglobulin Isotypes:

(IgG, IgM, IgA, IgE, IgD) refers to the genetic

variations or differences in the constant regions of the
heavy and light chains

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Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

Polyclonal antibodies:
• Derived from multiple B cell clones
• Recognize multiple epitopes on Inject with antigen
antigens

“epitope” = unique part of

Collect Blood Serum
antigen recognized by antibody
purify antibodies w/affinity
chromatography
using antigen attached to
beads
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Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

Monoclonal antibodies:
• Derived from B-cell clone
• Recognize single epitope on antigen

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Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

• Washing steps are necessary to remove unbound reagents and increase the signal:noise ratio.

• Insufficient washing allows high background

• Excessive washing decreases sensitivity (elution of the antibody)

• Washing buffer formulations consist of only a physiological buffer such as

phosphate buffered saline (PBS).
 a detergent such as 0.5% Tween 20 is added to help remove
nonspecifically bound material.

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Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

• The choice depends upon the species of animal in which the primary antibody was raised (the host species) or any
tag on that antibody (i.e., biotin or DIG).
 For example, if the primary antibody is an unmodified mouse monoclonal antibody then the secondary antibody
must be an anti-mouse IgG secondary antibody obtained from a non-mouse host

• Antibodies for Western blotting are typically used as dilute solutions, ranging from a 1:100-1:500,000 dilution from
a 1 mg/ml stock solution.

 Prepared in blocking solution (presence of detergent and blocking agent)

 Too much detergent and blocking agent may prevent efficient binding of the antibody and thus reducing
the signal.

 The optimal dilution of a given antibody with a particular detection system must be determined experimentally.

 Using lower amounts of antibody may reduce background because of its increased specificity for the target with
the highest affinity.
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Membrane Incubation with Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

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 Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

•Detection of HRP labeled secondary antibody by chemiluminescence

• Electrochemiluminescence (ECL) reagent: H2O2 + luminol

• HRP catalyzes breakdown of H2O2 to H2O and O2
• Luminol is oxidized
• Light from oxidized luminol is detected using film

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Membrane Incubation with Blocking Solution and Antibodies

Blocking Primary antibody Wash Secondary antibody Wash Detect

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 Protocol
(Step by Step)

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 Step
1 Gel Electrophoresis
PROTOCOL

The proteins of the sample are separated according to size on a gel

Sample Preparation
• Add lamellae sample buffer containing reducing agent (BME) to protein sample.
• Heat sample mixture at 95oC for 5 minutes.

Electrophoresis
• While protein samples are heating, assemble electrophoresis unit.
• Prepare Gels:
• We will be using 12% Acrylamide gels because our protein of interest (DJ-1) is 20 kDa in size.
• Load Gel
• Molecular weight marker (5-10µl) and protein samples (30µl).
• Run gel at 200V for 30 minutes.

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 Step
2 Membrane Transfer (Blotting)
PROTOCOL

• Soak PVDF membrane in Methanol for 10 sec

• Dilute in distilled water for 1min.
• When gel run is complete, set up the transfer apparatus as shown in the figure.
• Run transfer at 100V for 1 hour.

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Filter paper

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 Step
2 Membrane Transfer (Blotting)
PROTOCOL

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 Step
3 Blocking
PROTOCOL

•Block the membrane with blocking solution containing:

• 5% BSA or
• 5% milk
•Block the membrane for 45-90 minutes at RT with constant agitation

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 Step
4 Primary Antibody incubation and Washing
PROTOCOL

•Prepare Primary Antibody:

• Dilution depends on the antibody used
• Ex: Prepare a 1:500 dilution of primary antibody (Goat Anti-DJ1) in blocking
buffer.

•Incubate membrane in primary antibody solution overnight at 4C with gentle rocking

or at RT for 1hr.

•Membrane Washing: Wash membrane 3 x 5 minutes each in PBS-Tw with gentle

shaking at room temperature.

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 Step
4 Secondary Antibody incubation and Washing
PROTOCOL

•Prepare secondary Antibody:

• Dilution depends on the antibody used
• Ex: Prepare a 1:1000 dilution of secondary antibody (Donkey Anti-Goat IgG-
HRP) in blocking buffer.

•Incubate membrane in secondary antibody solution for 2hr at room temperature with
gentle shaking.

•Repeat Membrane Washing

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 Step
5 Detection
PROTOCOL

•Visualization of Protein of Interest

• add 1 ml of ECL solution B to 1 ml of ECL solution A, mix, and add directly to
membrane.
• Incubate in the dark for 3-5 minutes, remove excess solution, and place membrane
protein in a nylon folder.
• Develop film & identify protein of interest.

DJ-1 knockdown in SF cells

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 Drawbacks
&
Troubleshooting

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Some Drawbacks of Western Blotting

1. Many steps where errors can occur

2. Large amount of sample needed (5-50 mg)

3. Accurate quantitation is very difficult

4. Time consuming protocol

PDF: Western blot troubleshooting 33

Problems and Troubleshooting

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