Labeling cultured cells with 32P(i) and preparing cell lysates for immunoprecipitation

This unit describes 32P(i) labeling and lysis of cultured cells to be used for subsequent immunoprecipitation of proteins. The approach is appropriate, however, for labeling any cellular constituent with 32P. This procedure is suitable for insect, avian, and mammalian cells and can be used with both adherent and nonadherent cultures. The general approach involves biosynthetic labeling with 32P(i) in medium containing a reduced concentration of phosphate. This approach can also be modified to label any cellular constituents with 32P(i). The first procedure described is 32P(i) labeling of adherent or nonadherent (e.g., hematopoietic) cells with subsequent lysis in a detergent buffer. More rigorous lysis conditions to be used for working with proteins that are difficult to solubilize are also described.