Western Blot

Sample Preparation

Proteins are extracted from Cells or tissues using lysis buffers such as RIPA or NP-40. A protein extraction buffer containing detergents and protease inhibitors to prevent degradation and preserve Post Translational Modifications.

Electrophoretic Separation (SDS-PAGE)

Proteins are separated by SDS-PAGE based on their molecular weight. The SDS provides a net-negative charge to the proteins, allowing them to migrate through a sieve-like gel matrix toward a positive electrode.

Protein Transfer

Separated proteins are transferred from the gel onto an adsorbent membrane (PVDF or nitrocellulose membrane) using electric current called as electroblotting. An important step to preserves the protein pattern from the gel and making proteins accessible for antibody based detection.

Blocking

To prevent non-specific antibody binding, the membrane (PVDF or nitrocellulose membrane) is "blocked" with reagents like non-fat dry milk or bovine serum albumin (BSA)

Antibody Incubation

The membrane is incubated with a primary antibody specific to the target protein. A secondary antibody labeled with enzymes like HRP (horseradish peroxidase) or fluorescent tag binds to the primary antibody. This enables detection and Visualization of the targeted protein

Detection

Protein bands are visualized using chemiluminescence or fluorescence detection systems. It is captured on X-ray film or digital imaging systems to quantify the protein's abundance.

Western blotting (WB), also known as immunoblotting, is one of the most widely used techniques in molecular biology for the detection and analysis of specific proteins. The method combines gel electrophoresis and antibody-based detection to identify proteins within complex biological samples. Due to its sensitivity, specificity, and reliability, Western blotting has become a fundamental tool in research, diagnostics, and proteomics.

HISTORY

Long ago, in 1807, a scientist named Ferdinand Frederic Reuss noticed something strange and exciting. When he passed electricity through water mixed with tiny particles, the particles began to move as if they were alive and following an invisible path. He had unknowingly discovered the principle of electrophoresis.

Many years later, in 1955, another scientist named Oliver Smithies wanted to understand proteins. He created a method called starch gel electrophoresis. or the first time, scientists could see that proteins came in many different forms, almost like runners spreading out in a race.

Exciting year of 1979

George Stark, found a way to move proteins from a gel onto special paper using capillary action, almost like water soaking into a sponge.

Towbin used electricity to gently pull proteins from the gel onto a membrane called nitrocellulose. This method worked beautifully and became the foundation of the technique used today.

Principle

Western blotting is based on the principle of separating proteins according to their molecular weight through gel electrophoresis, transferring them onto a membrane, and specifically detecting the target protein using antigen-antibody interactions. It is not a one step technique.

Initially, proteins are separated by SDS-PAGE based on size. The separated proteins are then transferred onto a nitrocellulose or PVDF membrane through electroblotting.

To prevent non-specific binding, the membrane is blocked and subsequently incubated with a primary antibody specific to the target protein. A labeled secondary antibody binds to the primary antibody. This generates a detectable signal, such as chemiluminescence or fluorescence. The intensity and position of the bands help determine the presence, size, and relative expression level of the target protein.

BLOT DETECTION

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