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[52]Immunoprecipitation of proteins

1990; Academic Press; Linguagem: Inglês

10.1016/0076-6879(90)82054-6

1557-7988

Gary L. Firestone, Sandra D. Winguth,

Monoclonal and Polyclonal Antibodies Research

The discovery and use of fixed Staphylococcus aureus (Staph A) as an immunoadsorbent has been a major advance in routinely using antibodies as sensitive probes for selectively examining the expression of specific protein products from radiolabeled tissue. Many variations on the original Kessler Staph A procedure have been reported for the immunoadsorption of radiolabeled antigens. Besides employing fixed Staph A, the most common variation is to use a solid-state matrix, such as Sepharose or agarose, that is covalently modified with isolated protein A molecules. The immunoadsorption works with either serum or isolated antibody preparations. The optimal dilution and the volume of antibodies needs to be determined for a given cell sample and antibody preparation. If needed, the serum or antibodies should be diluted in phosphate-buffered saline (PBS) and not in a detergent-containing solution. During the procedure, samples of radiolabeled cell extracts, unlabeled cell extracts, antibodies, preimmune sera, Staph A, and BSA solutions should be kept on ice. The cellular fractions are more likely to contain radiolabeled protein which nonspecifically associates with the Staph A pellets. For secreted fractions, which contain significantly lower amounts of total nonspecific radiolabeled proteins, the second immunoadsorption step is not as critical.