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Methods Mol Biol. 2016;1360:47-58. doi: 10.1007/978-1-4939-3073-9_4.

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

Author information

1
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
2
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.
3
Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA. jinglese@mail.nih.gov.

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.

KEYWORDS:

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

PMID:
26501901
PMCID:
PMC4734380
DOI:
10.1007/978-1-4939-3073-9_4
[Indexed for MEDLINE]
Free PMC Article

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