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Anal Biochem. 2011 Oct 1;417(1):97-102. doi: 10.1016/j.ab.2011.05.046. Epub 2011 Jun 7.

A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases.

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  • 1Division of Gastroenterology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48105, USA. andrewwt@med.umich.edu

Abstract

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:
21704602
[PubMed - indexed for MEDLINE]
PMCID:
PMC3143478
Free PMC Article
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