Metabolic Labeling of Protein Antigens with [³²P]Orthophosphate

In this protocol, cells are cultured in the presence of [³²P]orthophosphate used by the cell to phosphorylate proteins. Cells must be maintained at 37°C at all times during this procedure. A drop in temperature will significantly reduce cellular metabolic activity and subsequently decrease the uptake of [³²P]orthophosphate. Once the cells are labeled, cell lysates are prepared for immunoprecipitation, followed by SDS-PAGE analysis. The gel can be dried directly or transferred onto a nitrocellulose or polyvinylidene fluoride (PVDF) membrane for analysis by western blotting and imaging by autoradiography or phosphorimager.