Western Blotting - Antigen Antibody Reactions

Western Blotting

Western blotting is called so because the procedure is similar to Southern blotting, which was developed by Edwin Southern for the detection of DNA. While Southern blotting is done to detect DNA, Western blotting is done for the detection of proteins.

      Western blotting is usually done on a tissue homogenate or extract. It uses SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis), a type of gel electro-phoresis to first separate various proteins in a mixture on the basis of their shape and size. The protein bands thus obtained are transferred onto a nitrocellulose or nylon membrane where they are “probed” with antibodies specific to the protein to be detected. The antigen–antibody com-plexes that form on the band containing the protein recog-nized by the antibody can be visualized in a variety of ways. If the protein of interest was bound by a radioactive anti-body, its position on the blot can be determined by exposing the membrane to a sheet of X-ray film, a procedure called autoradiography. However, the most generally used detec-tion procedures employ enzyme-linked antibodies against the protein. After binding of the enzyme–antibody conju-gate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appear-ance of a colored band at the site of the target antigen. The site of the protein of interest can be determined with much higher sensitivity if a chemiluminescent compound along with suitable enhancing agents is used to produce light at the antigen site (Fig. 14-16).