Western blot explained in details | Applications of western blot | CSIR NET

Name: Western blot explained in details | Applications of western blot | CSIR NET
Uploaded: 2023-10-17T07:32:09.000Z
Duration: 32 min 39 s

Understanding Western Blot: A Comprehensive Overview

What is Western Blot?

Western blot is a widely used technique in molecular biology and biochemistry labs for detecting specific proteins within a mixture or patient sample.

The method determines the presence of a target protein (the pink protein in this context) through antigen-antibody interactions.

Workflow of Western Blot

The workflow consists of three main steps:

Protein extraction from cells, tissues, or serum.

Separation of proteins on a gel.

Detection of proteins using antibodies.

Step-by-Step Breakdown

Step 1: Protein Extraction

Proteins are extracted using lysis buffer to create a protein mixture necessary for analysis.

The lysis buffer composition includes components that prevent protein degradation, such as protease inhibitors.

Step 2: Gel Electrophoresis

Extracted proteins are separated using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), which sorts proteins based on their molecular weight.

The gel is made from acrylamide and N,N'-methylenebisacrylamide, polymerizing through free radical mechanisms.

Gel Composition and Functionality

Different percentages of gels are used depending on the size of the target protein; larger proteins require lower percentage gels (e.g., 10%) while smaller ones need higher percentages (e.g., 15%).

Understanding Gel Structure

Stacking vs. Resolving Gels

SDS-PAGE consists of two parts: stacking gel (5% concentration with larger pores for concentrating proteins before separation) and resolving gel (12%-15% concentration with smaller pores for actual separation).

Charge and Movement in Gel

Protein Separation and Detection Techniques

Overview of Ion Movement in Gel Electrophoresis

The chloride ion moves quickly towards the positive electrode due to its negative charge and smaller size, followed by proteins coated with SDS, and lastly glycine ions which are positively charged at pH 6.8.

Stacking vs. Resolving Gel

In gel electrophoresis, proteins align like athletes at a starting line before entering the resolving gel, which has a different pH (8.8). This change affects the movement of ions and proteins.

Protein Migration Dynamics

At pH 8.8, glycine becomes negatively charged (anionic), allowing it to move rapidly through the gel alongside chloride ions, while protein migration is based on molecular weight.

Running the Gel

The gel is run at approximately 200 volts for about half an hour; monitoring is essential to determine when to stop once the dye front exits the gel.

Staining Techniques for Protein Visualization

After running, gels are stained with reagents like Coomassie Blue to visualize protein bands corresponding to different molecular weights; a molecular weight ladder aids in this identification.

Choosing Appropriate Stains

Various stains exist for protein detection: Coomassie Brilliant Blue, silver stain (detecting as low as 5–10 ng), Ponceau S (200 ng or higher), each with unique sensitivities and applications.

Western Blotting Process

Transferring Proteins from Gel to Membrane

Following separation on a gel, proteins are transferred onto a PVDF membrane using electroblotting under an electrical field that drives negatively charged proteins toward the positive electrode.

Blocking Non-Specific Binding

A blocking step prevents non-specific antibody binding on membranes using solutions like non-fat milk or BSA, ensuring antibodies target only specific proteins of interest.

Antibody Application for Detection

Primary antibodies are added overnight at 4°C; if the target protein is present, it will bind. Subsequent washing removes unbound antibodies before adding secondary antibodies that bind to primary ones.

Developing the Blot

Different strategies exist for blot development: enzyme-linked secondary antibodies produce color upon substrate reaction; chemiluminescent methods generate light signals; fluorescence-based detection uses tagged secondary antibodies for visualization.

Imaging Techniques

Western Blot Applications in Biochemistry

Overview of Western Blot Technique

The Western blot is a fundamental technique widely used in biochemistry labs for various applications, particularly in clinical and biomedical research.

Studying Cell Signaling Pathways

Western blotting can be utilized to study cell signaling pathways, where phosphorylation of proteins serves as an indicator of pathway activation.

By comparing unphosphorylated versus phosphorylated forms of proteins, researchers can assess the activity levels within specific signaling pathways, such as the MAPK pathway.

Example: MAPK Pathway Activation

Increased levels of phosphorylated AR (a protein involved in the MAPK pathway) indicate that the signaling pathway is activated when ligands are present.

The ratio of phosphorylated to unphosphorylated AR provides insights into the activation state of this particular signaling cascade.

Detecting Nuclear Receptor Translocation

Another application involves detecting nuclear receptors that translocate from the cytoplasm to the nucleus upon ligand binding using Western blot analysis.

By extracting both nuclear and cytoplasmic fractions and analyzing them via Western blot, one can observe changes in protein localization—specifically, an increase in nuclear fraction correlating with ligand presence.

Conclusion and Engagement