Detecting Protein Antigens in Sodium Dodecyl Sulfate-Polyacrylamide Gels

Abstract

In some cases, a native protein can be isolated in its pure form from cell lysates or tissue preparation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Antigens purified this way often induce good antibody responses. After electrophoresis, the band of protein of interest must be located in the gel. A variety of identification methods can be used, all of which are designed to avoid excessive fixation of the protein in the gel matrix. The choice of method depends partly on the abundance of the polypeptide. Three methods are commonly used: (1) staining side strips cut from the edge of the gel, (2) light staining of the gel itself, and (3) locating the band by radioactive labeling of the antigen. Staining strips of the gel cut from its sides avoids the need to fix the gel. When isolating abundant proteins that are well separated from other bands, staining side strips is a useful method. If the protein is not abundant or is located close to a contaminating band making a clean excision difficult, use one of the other staining methods. If the protein is reasonably abundant, then a light staining of the proteins in the gel with Coomassie Blue G will permit localization without fixing. Alternatively, the bands in the gel can be visualized by immersing the gel in sodium acetate or copper chloride. If the protein is radiolabeled with 125 I, 32 P, or 35 S, then use an autoradiogram as a template to excise the band of interest.