Western Blot Analysis

Presented By: Maryam Rasool

Ph.D. Pharmaceutics First Semester
 Introduction
• Western blotting (WB) or protein immunoblotting is a popular laboratory
technique to detect specific proteins from a cell or tissue sample.
• This technique is very powerful to detect the presence of a given protein in a sample, to assess
posttranslational modifications such as phosphorylation and to characterize protein
complexes.
• The name Western blotting resembles the name Southern blotting, a technique for DNA
detection and the detection of RNA is termed northern blotting.

Figure 1:Western Blotting (Proteins transfer onto the membrane)

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 Basic Principle
Western blotting relies on the electrophoretic
separation of proteins from a complex mixture
based on their mass, the transfer of these
proteins to a solid matrix, and the detection of
specific proteins of interest on the matrix using
antibodies.

Figure 1: Steps in Western Blotting

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Process Overview

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Brief descriptions of the steps in a
Western blotting
1. Sample Preparation
 Sample Collection and Type:

Possible sources: Whole cell extracts, tissue extracts, and subcellular fractions.
Tissue Samples: Require homogenization or sonication to ensure proper cell lysis.

 Cell Lysis:

Purpose: To release proteins from cells and solubilize them.

Conditions: Performed on ice with protease inhibitors and phosphatase inhibitors to prevent protein degradation and de phosphorylation.

 Lysis Buffer:

Adjust pH and ionic strength as needed.

The cell lysis buffer used in extraction should align with the target protein cellular localization.
Detergent-Free Lysis: Use homogenizer, sonication.
For nuclear extracts: Treat with endonuclease to break down DNA and reduce viscosity.

 Removal of Insoluble Debris:

Filter or centrifuge lysates to remove cellular debris before loading onto the gel. 5
 Determining Protein Concentration:
Essential for equal loading on the gel.
•Assays:
•BCA Assay
•Bradford Assay
•Lowry Assay
 All western blot samples have three elements: protein extract, cell lysis buffer, and Laemmli (sample) buffer.
 Laemmli buffer (60 mM Tris-HCl pH 6.8; 20% glycerol; 2% SDS; 4% beta-mercaptoethanol; 0.01% bromophenol blue) is
unique to western blot sample preparation as each reagent is purposeful for SDS-PAGE.

Figure 3: Sample collection

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 2. Gel Electrophoresis
Purpose:
Separation of proteins based on molecular weight.

Types of Gels:
Utilizes a gel with two layers: stacking gel (top) and separating/resolving gel (bottom).

Stacking Gel Separating (Resolving) Gel

Purpose: Align proteins into thin, sharply Purpose: Separate proteins based on size.
defined bands.

Properties: Properties:
• Slightly acidic pH (6.8). • Basic pH (8.8).
• Lower acrylamide • Higher polyacrylamide content:
concentration: Creates a more Narrows gel pores.
porous gel. • Separation by size: Smaller
• Poor at separating proteins by proteins move more rapidly than
size but allows proteins to align. larger ones.

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Figure 3: Illustration of Process of Protein Separation Based on MW

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PROCESS

 Pour the running buffer into the electrophorator.

 Place gel inside the electrophorator and connect to a power supply. Make sure buffer covers the gel completely, and
remove the comb carefully.

 Load marker (6 μL) followed by samples (15 μL) in to each well.

 Run the gel with low voltage (60 V) for separating gel; use higher voltage (140 V) for stacking gel.

 Run the gel for approximately an hour, or until the dye front runs off the bottom of the gel

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 3. Transfer to Membrane (Blotting)
 Overview:
Blotting is the electrophoretic transfer of proteins from a gel onto a membrane.
 Methods: (Electro blotting) Wet, and semi-dry.
 Principle: Proteins (negatively charged) migrate toward the anode in a transfer sandwich with a modified buffer.
 Key Component:
Towbin Buffer: Standard buffer for transfer.
• Composition: 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3.
• Role of Methanol: Increases protein hydrophobicity, releases SDS, enhancing protein adsorption onto the
membrane.
 Transfer Sandwich Setup:
Order (cathode to anode): Filter paper → Gel → Membrane → Filter paper.

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Figure 4: Transfer Sandwich Setup

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 Wet vs. Semi-Dry Transfer Systems

Wet Transfer Semi-Dry Transfer

Uses a large tank of transfer buffer. Only requires dampening of the transfer
Fiber pads (sponges) on each side for sandwich (low buffer volume).
consistent contact.

Time Requirement: Low voltage applied Time Requirement: Blotting completes

overnight within an hour.

Advantages: Advantages:
• High transfer efficiency across a • Time-efficient.
range of protein sizes. • Reduces buffer volume.
• Best for large proteins (e.g., Limitations:
membrane receptors). • Not ideal for large proteins.
• Cooling Effect: Methanol and • Lower transfer efficiency
cold buffer reduce overheating compared to wet transfer.

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Types of Membranes for Blotting

1. Nitrocellulose Membrane:
Features:
• High protein affinity and retention.
• Brittle, single-use.
Pore Size Options: 0.45 microns suitable for most proteins.
Shrinkage: Methanol can cause shrinkage; caution needed for large proteins.

2. PVDF (Polyvinylidene Difluoride) Membrane:

Features:
• High protein binding capacity, strong chemical resistance.
• Enables reprobing and storage.
• Enhanced Transfer Efficiency: Especially in the presence of SDS.
Considerations:
• Can increase background signal due to higher sensitivity.
Choice Based on Target Protein and Detection Method:
• Small pore sizes preferred for smaller proteins, but 0.45 microns works for most proteins.

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4. Blocking
Purpose:
• Prevents non-specific binding of antibodies to the
membrane.

Blocking Agents:
• Bovine Serum Albumin (BSA): Commonly used; non-
fat dry milk also effective.
• Commercial Blocking Buffers: Tailored for specific
applications.

Optimal Conditions:
• Blocking solution concentration (5-10%), incubation
time (1 hour to overnight), and temperature (room
temperature or 4°C).

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Washing
Between antibody incubation steps, the membrane must be washed with TBS + 0.1% Tween 20 (TBST) or PBS
+ 0.1% Tween 20 (PBST) to remove unbound antibodies and to reduce the background signal. While too little
washing can result in a high background signal, too much washing can reduce the signal-to-noise ratio.

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5. Antibody Probing
During the detection process the membrane is probed for the protein of
interest with antibodies, and links them to a reporter enzyme, which drives
a colorimetric or photometric signal.
For a variety of reasons, this traditionally takes place in a two-step
process, although there are now one-step detection methods available for
certain applications.

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Two-Step Antibody Incubation in Western Blotting

Primary Antibody
•Generation:
• Produced by immunizing a host species (e.g., rabbit, mouse) with the target protein, eliciting an immune response.
•Incubation:
• Dilution: 0.5 to 5 µg/mL in a buffered saline solution (e.g., PBS).
• Buffer Composition: Includes a small percentage of detergent (e.g., Tween-20) and blocking agents (e.g., powdered milk or BSA) to
minimize non-specific binding.
• Conditions: Incubate with the membrane under gentle agitation for 30 minutes to overnight at various temperatures; warmer
conditions may enhance both specific and non-specific binding.

Secondary Antibody
Purpose: Binds to the species-specific portion of the primary antibody to amplify the signal.
Linkage Options:
Commonly conjugated to:
Reporter Enzymes: Alkaline phosphatase (AP) or horseradish peroxidase (HRP).
Advantages:
Multiple secondary antibodies can attach to one primary antibody, enhancing detection sensitivity.

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One-Step Probing in Western Blotting
Historical Context
•Traditionally, Western blotting involved a two-step process (primary and secondary antibodies) for ease of production and flexibility.
•This method included an amplification step that enhances detection sensitivity.
Advantages of One-Step Probing
Speed and Efficiency:
One-step systems enable faster processing and reduced use of reagents.
Direct Detection:
Utilizes a single probe antibody that recognizes the protein of interest and carries a detectable label (e.g., biotin, enzyme).
Application:
Suitable for high-throughput protein analysis, especially when large quantities of antibodies are needed.
Process Overview
Incubation:
The labeled primary probe is incubated with the membrane, similar to the two-step process.
Washing Steps:
Unbound probes are washed away to prepare for detection.
Analysis
Detection of Bound Probes:
After washing, detection occurs directly based on the bound, labeled probes.
Size Estimation:
Bands are compared to a protein marker/ladder for size approximation.
Normalization:
Structural proteins (e.g., actin, tubulin) are analyzed alongside target proteins to control for loading variability and ensure accurate
quantification.

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6. Detection Methods in Western Blotting

1. Colorimetric Detection
•Enzymes Used:
• Commonly employs Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP).
• HRP is favored for its stability, cost-effectiveness, and versatility.
•Process:
• A chromogenic substrate is added to the membrane.
• The substrate undergoes a chemical change due to enzyme activity, producing a color change visible for imaging.
•Considerations:
• Prompt imaging is crucial as colors fade upon drying.
• Ideal for straightforward visualization of protein bands.

2. Chemiluminescent Detection
Enzymes Used:
Typically utilizes HRP for its efficiency.
Process:
A chemiluminescent substrate is added, producing light as the enzyme reacts.
Signal duration is short (1–24 hours), requiring timely detection.
Recording:
Signals are captured using X-ray film or digital imaging for permanent records.
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3. Fluorescent Detection
•Mechanism:
• Detection antibody is conjugated with a fluorophore.
• Excitation with specific light wavelengths causes the fluorophore to emit light at different
wavelengths.
•Imaging:
• Requires specialized digital imagers (e.g., APD, PMT, CCD cameras).
• No substrate step involved, which shortens the protocol.
•Multiplexing:
• Allows simultaneous detection of multiple proteins using different fluorophores.

4. Radioactive Detection
Historical Use:
Involves conjugating a radioisotope to the detection antibody.
Detection Method:
Emitted radiation is captured on X-ray film.
Limitations:
Requires special handling for safety, is expensive, and has a limited shelf life due to decay.
Mostly replaced by non-radioactive methods due to these constraints.

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The most common result of your experiment
 A good Western blot.
 Another possible result.
 No signal detected or weak signal.

Most likely reasons:

1. Antibody was not suitable (poor quality antibody).

2. Insufficient protein loaded on the gel for the amount of antibody used (or antibody too diluted).
3. Transfer efficiency from gel to membrane was poor.
2. ECL reagent was expired or contaminated.
5. Incorrect secondary antibody used.
6. Blocking agent concentration was too high.

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Chemiluminescent Western Blot

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 Protocol
Materials Needed:

•Protein samples (cell lysates or purified proteins)

•SDS-PAGE gel (precast or self-cast)
•Transfer buffer (e.g., Tris-Glycine buffer with methanol)
•PVDF or nitrocellulose membrane
•Primary antibody (specific to the protein of interest)
•Secondary antibody (conjugated to HRP or another detection enzyme)
•Blocking solution (e.g., 5% non-fat dry milk in TBST)
•TBST (TBS with Tween 20)
•Chemiluminescent substrate or other detection reagents
•Molecular weight markers (protein ladder)
•Electrophoresis system, blotting system, and imaging device

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1. Cell Collection
Place a 35 mm dish of cells on ice to inhibit protease activity, preserving target proteins.
Discard media and wash with 2 mL ice-cold phosphate-buffered saline (PBS) to remove any residual proteins from the culture
media.
Discard PBS thoroughly after the wash.

2. Cell Lysis
Add 100–500 μL NP-40 lysis buffer (prepared fresh with protease inhibitors) to the cells.
Gently lift cells using a cell scraper and transfer to 1.5 mL microcentrifuge tubes.
Incubate tubes on ice for 5–30 minutes to allow the plasma membrane to rupture and release proteins into the lysate.
Note: For challenging proteins, you may extend the incubation at room temperature (RT) to maximize yield.
Centrifugation
Centrifuge lysate at ≥12,000xg for 10 minutes at 4°C to separate cell debris (e.g., nuclei, membranes).
Carefully transfer the clear supernatant (containing target proteins) to a new tube.

3. Protein Quantification
• Quantify protein concentration using BCA, Bradford, or Lowry assays for accurate loading.
• Sample Preparation for Gel Loading
• Combine lysate with 2x sample buffer, which includes:
• 62.5 mM Tris-HCl (pH 6.8)
• 2% SDS (for protein denaturation)
• 25% glycerol (for sample weight and gel loading ease)
• 0.01% bromophenol blue (tracking dye)
• 1–5% β-mercaptoethanol or 100 mM DTT (reducing agents for disulfide bonds)
• Boil samples at 95°C for 5 minutes to denature proteins and inactivate enzymes.
• Cool on ice for 1 minute, then centrifuge at ≥12,000xg for 5 minutes at RT to remove aggregates.

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4. Gel Preparation
•Cast the resolving and stacking gels before starting:
• 10% Resolving Gel (10 mL): Separates proteins 10–70 kDa.
• 2.5 mL 40% Bis/Acrylamide mix (29:1)
• 5 mL 1.5 M Tris (pH 8.8)
• 2.5 mL MilliQ water
• Add immediately before casting: 12.5 μL 40% APS, 5 μL TEMED
• 4% Stacking Gel (10 mL): Concentrates proteins before resolving gel.
• 1.25 mL 40% acrylamide
• 0.65 mL 2% bis-acrylamide
• 1.25 mL 1 M Tris-HCl (pH 6.8)
• 6.85 mL MilliQ water
• Add immediately before casting: 25 μL 40% APS, 10 μL TEMED
• Pour gels carefully, covering the resolving gel with isopropanol during polymerization. Rinse with double distilled water before adding the
stacking gel.
5. Gel Loading
• Load 20–50 μg of protein per lane. Overloading can distort bands, so aim for the minimum amount for good detection.
• Load a protein ladder (preferably prestained) for monitoring and size reference.
6. Running the Gel
• Set gel to run in 1x TGS buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3):
• 50 V until proteins enter resolving gel, then increase to 100–150 V.
• Note: Monitor migration to prevent overheating, which distorts bands.
7. Electrotransfer to Membrane
• Pre-treat PVDF membrane with methanol for 5 minutes to activate it, then equilibrate in transfer buffer:
• 48 mM Tris, 39 mM glycine, 20% methanol.
• Equilibrate the gel in transfer buffer for 5 minutes.
• Assemble transfer “sandwich” with membrane between gel and anode.
• Run semidry transfer at 1 mA/cm² of membrane for 30–60 minutes.
• Verify transfer efficiency by staining the membrane with Ponceau S.

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8. Blocking
• Incubate membrane in 5% non-fat dry milk in TBST (20 mM Tris, 500 mM NaCl, 0.1% Tween-20, pH 7.4) for 15–30
minutes at RT to block nonspecific binding.
• For phospho-proteins, use 5% BSA in TBST to reduce background noise.
9. Primary Antibody Incubation
• Dilute primary antibody in blocking solution as per manufacturer’s instructions.
• Incubate membrane with primary antibody for 2 hours at RT or overnight at 4°C with gentle rocking.
• Tip: Store antibody solution at −20°C with 0.02% sodium azide for reuse. Azide must be washed out before HRP
detection.
10. Washing & Secondary Antibody Incubation
• Wash membrane 3x for 10 minutes with TBST to remove unbound antibody.
• Dilute HRP-conjugated secondary antibody in TBST as recommended, and incubate for 1 hour at RT.
• Secondary antibody should not be reused, as it is sensitive to degradation.
11. Enhanced Chemiluminescence (ECL) Detection
• Prepare ECL substrate (1:1 mixture of the two components) just before use.
• Drain excess liquid, apply ECL substrate evenly over the membrane, and incubate for 1–5 minutes.
• Blot membrane gently to remove excess substrate, then detect chemiluminescence using X-ray film or an imaging
system.
• Optional: Mark protein ladder bands on membrane for future reference

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ARTICLE 1

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References
• https://
www.protocols.io/view/detailed-western-blotting-immunoblotting-pr
otocol-yxmvmn2rng3p/v1
.
• https://pmc.ncbi.nlm.nih.gov/articles/PMC3456489/#:~:text=Western
%20blot%20is%20often%20used,a%20band%20for%20each%20prote
in
.
• Xing, X.; Wang, X.; He, W. Advances in research on tumor
immunotherapy and its drug development. J. China
Pharmaceut. 2021, U52, 10–19. [Google Scholar]
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