Abstract

WESTERN BLOTTING: AN UNIQUE TECHNOLOGY FOR DETECTION OF PROTEINS BY ANTIGEN-ANTIBODY INTERACTION

Girish M. Kapadiya, Ashish M. Parmar and Dr. Dhrubo Jyoti Sen*

ABSTRACT

Western blotting is a widely used technique for the detection and analysis of proteins based on their ability to bind to specific antibodies. It was 1 st described by Towbin, et.al in 1979 and has since become one of the most commonly used methods in life science research. Western blotting is an accomplished rapidly, using simple equipment and inexpensive reagents; it is commonly used laboratory technique. The specificity of the antibody-antigen interaction enables to a target protein to be identified in the midst of a complex protein mixture. It is an analytical method where in a protein sample is subjected to electrophoresis on an SDS-PAGE (Sodium Dodecyl Sulfate-Poly Acrylamide Gel Electrophoresis) and electro transferred on to PVDF (Poly Vinylidene Fluoride) membrane or nitrocellulose membrane. The transferred protein is detected using specific primary and secondary enzyme labeled antibody. Antibodies bind to specific sequences of amino acids, known as the epitope. An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, β cells or T cells. For example, the epitope is the specific piece of the antigen that an antibody binds to. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes. The epitopes of protein antigens are divided into two categories, conformational epitopes and linear epitopes, based on their structure and interaction with the paratope. The paratope is the part of an antibody which recognizes an antigen, the antigen-binding site of an antibody. It is a small region (of 15–22 amino acids) of the antibody's Fv region and contains parts of the antibody's heavy and light chains. The part of the antigen to which the paratope binds is called an epitope. This can be mimicked by a mimotope. A mimotope is a macromolecule, often a peptide, which mimics the structure of an epitope. Because of this property it causes an antibody response similar to the one elicited by the epitope. An antibody for a given epitope antigen will recognize a mimotope which mimics that epitope. Mimotopes are commonly obtained from phage display libraries through biopanning. Vaccines utilizing mimotopes are being developed. The engraved inner portion of idiotype is the paratope where the epitope of the antigen binds. A conformational epitope is composed of discontinuous sections of the antigen's amino acid sequence. These epitopes interact with the paratope based on the 3-D surface features and shape or tertiary structure of the antigen. The proportion of epitopes that are conformational is unknown. By contrast, linear epitopes interact with the paratope based on their primary structure. A linear epitope is formed by a continuous sequence of amino acids from the antigen. Because amino acid sequences are different from protein to protein, antibodies can recognize specific proteins among a group of many. Therefore, a single protein can be identified in a cell lysate that contains thousands of different proteins and its abundance quantified through western blot analysis. First, proteins are separated from each other based on their size. Second, antibodies are used to detect the protein of interest. Finally, a substrate that reacts with an enzyme is used to view the antibody/protein complex.

Keywords: SDS-PAGE, PVDF, Nitrocellulose, Elisa, Epitope, Mimotope, Paratope, Gel electrophoresis, Chemiluminescence, Western Blotting, Antibody, Antigen