WesternBlot

Western blot, also known as immunoblotting, is a widely used analytical technique in molecular biology to detect specific proteins in a complex sample. It combines gel electrophoresis, which separates proteins by size, with immunoassay techniques, which detect specific proteins. The process begins with separating proteins from a biological sample, such as cell or tissue extract, based on their molecular weight using gel electrophoresis. The separated proteins are then transferred from the gel onto a solid support membrane, typically nitrocellulose or PVDF, to make them more accessible for antibody detection.

Following transfer, the membrane is blocked with a protein solution to prevent non-specific binding of antibodies. The membrane is then incubated with a primary antibody that is specifically designed to bind to the target protein of interest. After washing away unbound primary antibody, a secondary antibody conjugated to an enzyme or a fluorescent marker is added. This secondary antibody binds to the primary antibody. Finally, a detection reagent is added that reacts with the enzyme or fluorescent marker, producing a detectable signal, such as a light emission or a colored precipitate, at the location of the target protein on the membrane. The intensity of the signal is proportional to the amount of the target protein present in the original sample. Western blotting is crucial for confirming protein expression, identifying protein modifications, and quantifying protein levels in various biological research and diagnostic applications.