A rapid assay for protein kinases phosphorylating small polypeptides and other substrates

A new and rapid method of protein kinase assay is presented which is suitable for low-molecular-weight substrates, irrespective of their electrophoretic or chromatographic mobility. It depends on the phosphorylation of the substrates with [gamma-32P]ATP, hydrolysis of the pyrophosphate bonds by boiling in 1 N HCl, extraction of 32P with isobutanol-benzene, and measurement of the radioactivity of 32P-labeled phosphoesters in the water phase. The method is shown to be suitable for both tyrosine- and serine-phosphorylating protein kinases.