What is western blotting and how does it work?
Western blotting—or immunoblotting—is a technique used to detect, characterize and quantitate proteins. The process first involves the electrophoretic separation of a mixture of proteins, including the protein of interest, on a polyacrylamide gel. The separated proteins are then transferred or blotted to a nitrocellulose or a PVDF membrane to immobilize the protein. The protein of interest is then detected by a simple antigen-antibody reaction using appropriate antibodies. A primary antibody is used to bind to the specific protein of interest followed by a secondary antibody to detect the antigen-antibody complex. The protein of interest is visualized as bands on the membrane.
Western blotting applications
Using western blotting, proteins can be characterized for several aspects, including their presence or absence, abundance, phosphorylation status or localization. In addition to these qualitative aspects, western blotting can also be used for relative quantitation of proteins based on the band intensity between experimental and control samples.
