![]() ![]() At first, the protein samples are electrophoresed on SDS-PAGE. The western blotting technique is mainly used to identify a specific protein in a complex mixture along with the determination of its molecular weight. Western Blotting Principle – How does western blot work? To learn the technique of Western Blotting for the detection of a specific protein. Now these secondary antibodies are visualized by using different methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein. After that the secondary antibodies are added to it which will bind with the specific primary antibodies.These separated proteins are transferred or blotted onto a matrix generally known as nitrocellulose or PVDF membrane.At first the proteins of a given sample are separated by using SDS-polyacrylamide gel electrophoresis (SDS-PAGE).The western blotting is completed in three steps such as separation of protein by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize.Western blotting is also known as protein immunoblot because an antibody is used to specifically detect its antigen.This technique exploits the inherent specificity of antigen-antibody interaction to identify specific antigens by polyclonal or monoclonal antibodies. In molecular biology Western blotting is a rapid and sensitive assay for detection and characterization of proteins. ![]()
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