Question

Explain the working principle of western blotting and mention its uses

Answer 1

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The term “blotting” refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Southern blot is used for transferring DNA, Northern blot for RNA and Western blot for Protein. Western blotting (also called immunoblotting, because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis.Western blotting can produce qualitative and semi-quantitative data about the protein of interest. It is an important technique used in cell and molecular biology. It enables the researchers to identify the specific protein from mixture of proteins extracted from cells as well as evaluation of their size and amount. The SDS PAGE technique is prerequisite for western blotting.

PRINCIPAL

Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay for detection and characterization of proteins. It is based on the principle of immunochromatography where proteins are separated into polyacrylamide gel according to their molecular weight.

The protein thus separated are then transferred or electrotransferred onto nitrocellulose membrane and are detected using specific primary antibody and secondary enzyme labeled antibody and substrate.

USES

• It is most sensitive and specific test for determining size and amount of protein present in any material.

• The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample.

• A western blot is also used as the definitive test for Creutzfeldt-Jakob Disease, Lyme disease,  Hepatitis B infection and HSV-2 (Herpes Type 2) infection.

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