Immunoblotting.

Abstract

Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The detection of the protein of interest relies on the binding of an antibody that specifically recognizes the protein of interest exposed on the membrane. The protein of interest can be purified or mixed with other proteins as in cell or tissue extracts. Usually immunoblotting combines the resolution of proteins by gel electrophoresis with immunochemical detection and is referred to as "western blotting." Immunoblotting can be used to determine the presence and the steady-state level of the protein of interest in the sample, its relative molecular weight, and the distribution of the protein between cellular fractions. Immunoblotting can be performed using the antibodies raised against synthetic peptide antigens modified to mimic posttranslational modifications of proteins, such as phosphorylation and acetylation, to study these modifications in the protein of interest in vivo. When antibodies against the protein of interest are not available, immunoblotting can be performed using antibodies that specifically recognize the recombinant epitope tags (hemagglutinin [HA]-, Flag-, cMyc-, or glutathione-S-transferase [GST]) fused to the protein of interest using recombinant DNA techniques. Immunoblotting has a variety of research, clinical, and forensic medicine applications. It is also one of the standard techniques for characterization of antibodies from different samples of polyclonal sera or hybridoma supernatants.