In vitro protein kinase activity measurement by flow cytometry |
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Authors: | Bernsteel Donald J Roman David L Neubig Richard R |
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Institution: | a Department of Pharmacology, University of Michigan, 1301 MSRB III, Ann Arbor, MI 48109, USA b Department of Internal Medicine (Cardiovascular Medicine), University of Michigan, Ann Arbor, MI 48109, USA |
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Abstract: | 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-μ, and Akt) on multiple substrates simultaneously. We also discuss conditions necessary to optimize measurement of the activity of several kinases in a single sample. |
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Keywords: | Akt Protein kinase A Protein kinase C Flow cytometry Kinase Phosphorylation Detection |
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