Coimmunoprecipitation is most commonly used to test whether two proteins of interest are associated in vivo, but it can also be used to identify novel interacting partners of a target protein. In both cases, the cells, which may have been labeled with [35S]methionine, are harvested and lysed under conditions that preserve proteinprotein interactions. The target protein is specifically immunoprecipitated from the cell extracts, and the immunoprecipitates are fractionated by SDS-PAGE. Coimmunoprecipitated proteins are detected by autoradiography and/or by western blotting with an antibody directed against that protein. The identity of interacting proteins may be established or confirmed by Edman degradation of tryptic peptides. This protocol was used to identify pVHL-associated proteins. Conditions should be optimized for the protein of interest. This protocol was provided by Peter D. Adams (Fox Chase Cancer Center, Philadelphia, Pennsylvania) and Michael Ohh (Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts).
A CAUTION O RECIPE
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