What is the Western Blot Test?

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Western blot test, also called immunoblot, is a procedure for detecting antibodies to specific antigens in a sample of human or animal serum. It is a variant of immunoelectrophoresis and involves separating proteins by size in a gel and then moving the separated protein mixture onto a PVDF or nitrocellulose membrane that tightly binds protein molecules. The membrane is then probed with antibodies that bind to the antigen of interest, and the color generated by bound antibody provides a way to identify the presence of the antigen. This link bosterbio.com

Proteins are first electrophoresed on a polyacrylamide gel, and a specific lane is reserved for a protein marker (ladder) with a known molecular weight. When an electric current is applied, proteins separate into bands of different sizes due to their differing electrophoretic mobilities. The ladder band is then used to estimate the protein molecular weight by comparison with the other bands on the gel.

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The sample is then transferred from the gel to a nitrocellulose or PVDF membrane that has been coated with a blocking buffer, such as bovine serum albumin (BSA). The membrane is then exposed to a solution of primary antibodies that bind to the target protein and a secondary antibody that binds to the Fc region of the primary antibody. A substrate for the label antibody, usually horseradish peroxidase (HRP), is added, and if the antibody has bound to its target protein a signal is produced that can be detected by an imaging system.


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