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Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase

  • Mindy I. Davis
  • Douglas S. Auld
  • James Inglese
Part of the Methods in Molecular Biology book series (MIMB, volume 1360)

Abstract

Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.

Key words

Quantitative high-throughput screening (qHTS) Yes1 Glucokinase PI5P4Kα Kinase Bioluminescence Luciferase ADP-Glo 

Notes

Acknowledgement

This work was supported by the Molecular Libraries Initiative of the NIH Roadmap for Medical Research. The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Mindy I. Davis
    • 1
  • Douglas S. Auld
    • 2
  • James Inglese
    • 1
  1. 1.Division of Preclinical Innovation, National Center for Advancing Translational SciencesNational Institutes of HealthRockvilleUSA
  2. 2.Center for Proteomic ChemistryNovartis Institutes for Biomedical ResearchCambridgeUSA

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