In vitro protein kinase activity measurement by flow cytometry

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• 作者：Donald J. Bernsteel ; David L. Roman ; Richard R. Neubig
• 关键词：Akt ; Protein kinase A ; Protein kinase C ; Flow cytometry ; Kinase ; Phosphorylation ; Detection
• 刊名：Analytical Biochemistry
• 出版年：2008
• 出版时间：15 December 2008
• 年：2008
• 卷：383
• 期：2
• 页码：180-185
• 全文大小：258 K

文摘

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.