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.
Publication types
-
Research Support, N.I.H., Extramural
MeSH terms
-
Adaptor Proteins, Signal Transducing / metabolism
-
Adenosine Diphosphate / analysis
-
Adenosine Triphosphate / analysis
-
Glucokinase / analysis*
-
Glucokinase / metabolism
-
High-Throughput Screening Assays / instrumentation
-
High-Throughput Screening Assays / methods*
-
Humans
-
Indicators and Reagents
-
Luciferases, Firefly / metabolism
-
Luminescent Measurements / instrumentation
-
Luminescent Measurements / methods*
-
Phosphotransferases (Alcohol Group Acceptor) / analysis*
-
Protein Binding
-
Proto-Oncogene Proteins c-yes / analysis*
-
Substrate Specificity
Substances
-
Adaptor Proteins, Signal Transducing
-
GCKR protein, human
-
Indicators and Reagents
-
Adenosine Diphosphate
-
Adenosine Triphosphate
-
Luciferases, Firefly
-
PIP4K2A protein, human
-
Phosphotransferases (Alcohol Group Acceptor)
-
Glucokinase
-
Proto-Oncogene Proteins c-yes
-
YES1 protein, human