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39.2 Identification and purification of proteins

Antibodies can be used to identify and purify specific proteins from a mixture. Two most commonly used methods are Immunoprecipitation and immuno-affinity chromatography. Western blot is another commonly used technique to determine the presence and size of a specific protein.

39.2.1 Immunoprecipitation assay:

Immunoprecipitation is a method in which a specific antigen is pooled from a mixture by using a specific antibody. The antibody is added to a mixture of antigen along with staphylococcal protein A or G. The Fab portion of the antibody binds to the antigen or protein and Fc protein of the same antibody is captured by staphylococcal protein A or G beads. Unbound antibodies are removed by washing the beads with detergents. Specific antigens or protein bound with the antibodies are detached with the help of a strong denaturating agent (sodium dodecyl sulfate) and separated using a sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) gel.

Figure 39.2 Schematic representation of immunoprecipitation assay :