Immunoblot commonly referred to as “Western blot” (WB), is used to detect antibodies to specific epitopes of electrophoretically separated subspecies of antigens. Western Blotting or immunoblotting is to determine the relative amounts of the protein present in different samples, with a specific primary antibody. Development of electrophorteic transfer to a suitable membrane, by Towbin and others (Western Blot) now permits the rapid, efficient and quantitative transfer of protein and nucleic acids to immobilizing media.

In these assays, antigens are first electrophoresed and then blotted onto a piece of filter paper. Thus the filter paper is the solid phase to which the antigen is bound. The filter is

blocked and then reacted with appropriate tagged antibodies and then substrate. Spots can then be detected by immuno blotting. A color reaction indicates a positive test. These spots indicate the position of the protein of interest.

The transfer of electrophoretically resolved DNA, RNA, and protein to an immobilizing paper or membrane matrix has become a standard procedure for the sensitive and specific detection of biologically interesting macromolecules. Exact and reproducible replicas of separations are captured on the paper or membrane sheets. After transfer, antigens, antibodies, hormones, receptor proteins, and glycoproteins can be detected by staining or by specific fluorescent (FIA), enzyme-substrate (EIA), or autoradiographic (RIA) assay.

Western blots allow investigators to determine the molecular weight of the protein and to measure the relative amounts of protein present in different samples.

Western Blotting systems are useful for the transfer of protein samples from SDS-PAGE gel to nitrocellulose membrane support with gel cassette at high current. The cassette is submerged in a buffer tank in between anode and cathode cells.