A High-Throughput Radiometric Kinase Assay
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 wordsKinase Kinase assay Kinase inhibitor High-throughput screen Dot blot kinase assay Insulin receptor
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.).
- 1.Duong-Ly KC, Peterson JR (2013) The human kinome and kinase inhibition. Curr Protoc Pharmacol. Chapter 2:Unit 2.9. doi: 10.1002/0471141755.ph0209s60
- 2.Goldstein DM, Gray NS, Zarrinkar PP (2008) High-throughput kinase profiling as a platform for drug discovery. Nat Rev Drug Discov. 7:391-397. doi: 10.1038/nrd2541
- 3.Anastassiadis T, Deacon SW, Devarajan K et al (2011) Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol. 29:1039-1045. doi: 10.1038/nbt.2017
- 4.Davis MI, Hunt JP, Herrgard S et al (2011) Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol. 29:1046-1051. doi: 10.1038/nbt.1990
- 5.Karaman MW, Herrgard S, Treiber DK et al (2008) A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol. 26:127-132. doi: 10.1038/nbt1358
- 6.Fabian MA, Biggs WH, Treiber DK et al (2005) A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol. 23:329-336. doi: 10.1038/nbt1068
- 7.Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc. 1:968-971. doi: 10.1038/nprot.2006.149
- 8.Huebner VD, Matthews HR (1985) Autoradiography of gels containing 32P. Methods Mol Biol. 2:51-53. doi: 10.1385/0-89603-064-4:51
- 9.Zouboulis CC, Tavakkol A (1994) Storage phosphor imaging technique improves the accuracy of RNA quantitation using 32P-labeled cDNA probes. Biotechniques 16:290–292, 294Google Scholar
- 10.Palfi A, Hatvani L, Gulya K (1998) A new quantitative film autoradiographic method of quantifying mRNA transcripts for in situ hybridization. J Histochem Cytochem. 46:1141-1149. doi: 10.1177/002215549804601006
- 11.Anastassiadis T, Duong-Ly KC, Deacon SW et al (2013) A highly selective dual insulin receptor (IR)/insulin-like growth factor 1 receptor (IGF-1R) inhibitor derived from an extracellular signal-regulated kinase (ERK) inhibitor. J Biol Chem. 288:28068-28077. doi: 10.1074/jbc.M113.505032