Use of sequential immunoprecipitation to reveal discrete, separable populations of SV40 T-antigen binding to host cellular proteins.

Sequential immunoprecipitations were carried out to determine the usefulness of this method for separating subpopulations of SV40 T-antigen complexed to various combinations of the cellular growth regulatory proteins pRB, p107, and p53. This approach was used successfully to separate discrete populations of SV40 T-antigen in a quatramolecular complex with pRB, p53, and p107, a trimolecular complex with pRB and p107, and a trimolecular complex with p107 and p53. This method was used as the first step towards isolating T-antigen for subsequent phosphopeptide mapping to address whether alterations in the overt phosphorylation of this viral oncoprotein is a major determinating factor to separation of T-antigen populations by complexing with different combinations of cellular growth regulatory proteins.