Kinase activity-tagged western blotting assay

Determining cellular activities of protein kinases is a fundamental step for characterizing pathophysiological cell signaling pathways. Here, we optimized a non-radioactive method that detects protein kinases in tissues or cells after separation by SDS-PAGE and transfer onto polyvinylidene fluoride membranes. The method, kinase activity-tagged western blotting (KAT-WB), consists of five steps: electrophoresis of cell extracts that contain protein kinases, electroblotting proteins onto polyvinylidene fluoride membrane, denaturation-renaturation, phosphorylation, with or without an added substrate protein and immunodetection using anti-phospho-specific antibodies. KAT-WB detected autophosphorylation of one Tyr-kinase and site-specific phosphorylation of added substrate by multiple kinases. KAT-WB assay enables us to interrogate multiple kinase signaling pathways without using radioactive ATP. METHOD SUMMARY Kinase activity-tagged western blotting is designed to separate and detect multiple kinases that phosphorylate themselves or a specific substrate. Kinases on the blot are subjected to denaturation with guanidine and then stepwise renaturation, prior to phosphorylation using nonradioactive ATP and detection with phosphospecific antibodies.