Western blot

Importance of Western Blot

To detect a specific protein in a sample using western blot technique.

Western Blot Method

Electroblotting of proteins into nitrocellulose membrane.

Western Blotting Technique: Learning Objectives for Students

• <p>To identify the theory behind western blot technique.

• To fully comprehend the Western blot steps.

• To design a complete western blot experiment.

• To analyze the visualized protein and understand Western blotting application.</p>

Western Blot Experiment: Theoretical Background

A blot, in molecular biology, is the technique that involves the transfer of DNA, RNA and proteins from a gel to a membrane (nitrocellulose or PVDF). Proteins can be detected by western blot (immunoblot). Labeled probes or antibodies can bind blotted proteins and reveal them, making it easier to study them.

The Difference between Elisa and Western Blot

The ELISA test uses enzymes or antibodies attached to a solid surface to create the test surface. A sample is then added to the test surface. Antibodies or enzymes that react or attach to proteins indicate a positive result. The Western blot test is performed after gel-electrophoresis. The separated proteins are transferred (or blotted) onto nitrocellulose or nylon membranes, which are then identified by specific antibodies that are tagged by a secondary protein. It detects viral antigens (proteins usually on the surface of viruses) using antibodies against those proteins. A positive Western blot indicates viral antigen presence.

Western Blot Experiment Principle of Work

Western blot is a commonly used technique designed to study a specific protein in a sample containing many other proteins. Proteins in the sample are extracted then separated by gel electrophoresis (SDS-PAGE) according to their molecular weight. Then, the proteins are electrophoretically transferred (blotted) to a more durable surface (e.g. nitrocellulose or PVDF membrane). Finally, a specific antibody is used to detect the protein of interest. The bound and labeled protein can be visualized giving information about its molecular weight and relative quantity.