Identification of associated proteins by coimmunoprecipitation

Many protein-protein associations that exist within the cell remain intact when a cell is lysed under nondenaturing conditions. Thus, if protein X is immunoprecipitated, then protein Y, which stably associated with X, may also precipitate. Coimmunoprecipitation is most commonly used to test whether two proteins of interest are associated in vivo, but it can also be used to identify interacting partners of a target protein. In both cases, the cells, labeled with [³⁵S]methionine, are collected and lysed under conditions that preserve protein-protein 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. Some early examples of this method include the use of antibodies to viral antigens to determine the host cellular proteins that interact with these viral transforming oncoproteins. Two interacting proteins of particular note are the tumor suppressor proteins p53 and pRB^1,2,3. This protocol was used to identify pVHL-associated proteins; conditions should be optimized for the protein of interest.