Immunoprecipitation for Antigen Localization

Immunoprecipitation was introduced by Schwartz and Nathenson (1 ) who used the procedure to isolate radioactively labeled antigens extracted by nonionic detergent. The procedure selectively precipitates an antigen(s) of interest using antibodies as a specific selection component of a precipitating complex. In this way the antigens are isolated and resolution and detection of the antigen by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) is improved by removal of other cellular components. A notable feature of immunoprecipitation protocols used in malaria research is the wide variety of assay conditions used. This reflects the robust nature of this procedure in that antibody to antigen complexes form readily under a variety of conditions. Immunoprecipitation protocols have usually followed the following for-mat: antigens are liberated from parasites by extraction or solubilization with detergent to give a total cellular extract. The soluble extract containing an antigen of interest is incubated with a specific antibody, or perhaps a polyspecific antibody, and a complex of antigen and antibody forms. The complex is sedimented by centrifugation following binding of protein A-Sepharose or fixed Staphylococcus aureus cells (2 ) to the Fc por-tion of the antibody. Alternatively, antigen can be sedimented directly by binding of specific antibody conjugated directly to chromatography beads (3 ) Precipitated antigens are separated by SDS-PAGE or two-dimensional electrophoresis. Antigens are then detected either by autoradiography for metabolically labeled antigens or conventional immunoblot analysis for non-radioactive antigens.