In vitro protein kinase activity measurement by flow cytometry.
Protein kinases are important drug targets, and a wide variety of methods have been developed for assessing their activity. A key element in developing selective kinase inhibitors is the ability to rapidly compare the effects of an inhibitor on several related or unrelated kinases. We describe a simple, nonradioactive, bead-based method for detecting kinase activity in vitro. Biotinylated peptide substrates are immobilized on beads and phosphorylation is detected with anti-phosphopeptide antibodies with no separation steps required. Phosphorylation is dependent on the amount of kinase in the assay and can be inhibited by known kinase inhibitors in a concentration-dependent manner. Using Luminex technology, we measured the activity of three kinases (PKA, PKC-mu, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample.
Bernsteel
Donald J
DJ
Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109, USA.
Roman
David L
DL
Neubig
Richard R
RR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005434:Flow Cytometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity; D054460:rho-Associated Kinases / Q000378:metabolism
D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D054460:rho-Associated Kinases; D019311:Staurosporine