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A High-Throughput Radiometric Kinase Assay

  • Krisna C. Duong-Ly
  • Jeffrey R. Peterson
Part of the Methods in Molecular Biology book series (MIMB, volume 1360)

Abstract

Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening of libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small-molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases.

Key words

Kinase Kinase assay Kinase inhibitor High-throughput screen Dot blot kinase assay Insulin receptor 

Notes

Acknowledgements

We would like to thank Dr. Jinhua Wu and Dr. Stevan Hubbard for providing insulin receptor kinase baculovirus and for advice on expression and purification of the kinase. The authors’ work is supported by NIH R01 GM083025 (J.R.P.), NIH R21 AI099929 (J.R.P.) and NIH T32 CA009035-36 (K.C.D.).

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Cancer Biology ProgramFox Chase Cancer CenterPhiladelphiaUSA

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