Permeabilization strategies to study protein phosphorylation

This unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. These experiments generally utilize [g-(32)P]ATP as an exogenously added phosphate donor, although [g-(32)P]GTP can be used in specific cases. Procedures are outlined for performing a protein phosphorylation experiment using permeabilized cells and isolated intracellular organelles. Both of these procedures result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane-bound kinases. Two different methods for determining the specific radioactivity of (32)P -containing compounds are also included.