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A fluorescence polarization assay for native protein substrates of kinases
- Source :
-
Analytical Biochemistry . May2003, Vol. 316 Issue 1, p41. 9p. - Publication Year :
- 2003
-
Abstract
- Protein phosphorylation is the mediator of many important cellular processes of signal transduction and cell regulation. Phosphorylation often occurs on multiple sites within a single protein, whereby the results of individual phosphorylations are not well defined. This is partially due to the lack of tools for analyzing specific phosphorylation states in a quantitative manner. We have developed a high-throughput, rapid, and quantitative method for the determination of the phosphorylation status of peptides and, more importantly, native protein substrates of kinases using a competitive fluorescence-based approach. We have applied our method to measuring the phosphorylation activity of the Wee1 and Myt1 kinases. Our technique allows one to monitor the bis-phosphorylation status of the Cdk2 protein using an antibody specific for bis-phosphorylated Cdk2 and a fluorescently labeled bis-phosphorylated Cdk2 peptide. We have used this assay to screen a library of 16 general kinase inhibitors against Wee1 and Myt1 activity. None of the inhibitors inhibited Wee1, but both staurosporine and K-252a inhibited Myt1, with <f>IC50</f> values of <f>9.2±3.6</f> and <f>4.0±1.3 μM</f>, respectively. [Copyright &y& Elsevier]
- Subjects :
- *PHOSPHORYLATION
*PROTEIN kinases
Subjects
Details
- Language :
- English
- ISSN :
- 00032697
- Volume :
- 316
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Analytical Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 9495857
- Full Text :
- https://doi.org/10.1016/S0003-2697(03)00033-2