Western Blot Protocol

Western Blotting

By applying the method for Southern blotting, developed by Dr. Edwin Southern in the 1970's, Towbin subsequently created the Western blot, with the term transfer coining the term "blot." (2) Very small quantities of a specified protein in a cell or in a body fluid can be detected by the Western blotting immunoassay technique. This technique begins with the sample of interest being electrophoresed on an SDS-PAGE, which
will resolve the proteins on a gel. The protein mixture is subjected to SDS, sodium dodecyl sulphate, an anionic detergent, that disrupts non-covalent bonds and denatures proteins. These coated proteins contain a net negative charge, which is proportional to their molecular weight. Electrophoresis of these protein subunits within an electric gradient can separate them based on mass. The separating gel, is formed from polymerizing and cross-linking acrylamide molecules. Moreover, a plot of protein mobility versus log molecular weight usually yields a straight line, from which unknown band molecular weight can be found. (1,2,4).

The proteins, now separated, can be transferred, through blotting, by contacting the SDS-PAGE gel to a polymer sheet, such as nitrocellulose, on which they stick non-specifically and irreversibly. An electric field applied at a right angle to the sheet, can separate the proteins, which now migrate horizontally out of the gel and onto the nitrocellulose, maintaining and fixing the pattern of their SDS-PAGE separation on the nitrocellulose. After, incubation with non-specific, excess, irrelevant protein to block any left over non-specific sites on the nitrocellulose sheet, the blot can now become a solid "antigen" support for further probing with specific antibodies. The antibodies only bind to their respective antigens, if these antigens are present within the bands on the nitrocellulose sheet. Followed by washing, any antibodies complexed to their antigens are revealed by staining.
Staining can occur with numerous different reagents, such as labeled secondary antibodies or Protein G, which is a molecule from bacteria that binds to the Fc portion of antibodies, both of which are usually labeled by a radioisotope or an enzyme specific for cleaving a chromogenic substrate.

A major requirement for a primary antibody (blotting antibody) to work effectively in a Western blot experiment, the use of antibodies that bind to epitopes that are not denatured during the SDS binding process. Epitopes can be conformational, linear and newly created by limited proteolysis. For example, a conformational determinant, through denaturation upon SDS application, the determinant (epitope) is lost by the denaturation process. Moreover, linear determinants, can be accessible or inaccessible depending on denaturation. Antibody can bind to determinant in both native and denatured states, while other epitopes can only be bounds in denatured proteins only. Therefore, our antibodies of interest many not react with denatured epitopes. Theerefore, we can utilized polyclonal antisera to increase the chance that some epitopes may survive the denaturation process, for a poorly defined protein. More specifically, it would be advisable to use an antibody, a monoclonal, that we know binds to a denatured epitope (or one that is not altered after denaturation). The use of a monoclonal antibody would decrease, reduce any background cross-reactivity. (1)

References:

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing. 4th ed., New York, USA, pages 1-40, 2.5-2.22, 3.1-3.12.

2. Delves, P. and Roitt, I. 1999. Encyclopedia of Immunology: Academic Press Inc., 2nd ed., San Diego, USA page 238.

3. 1994. Current Protocols in Molecular Biology. Volume 2. John Wiley & Sons Inc., USA

4. Cruse, J. and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA pages 1960-1965.