A scintillation proximity assay for studying inhibitors of human tau protein kinase II/cdk5 using a 96-well format.
Dysregulation of the brain-specific tau protein kinase II (TPK II)/cdk5 is reported to play an important role in the pathogenesis of Alzheimer's disease. We report here a quantitative scintillation proximity assay (SPA), which is suitable for determining TPK II/cdk5 activity and its inhibition. It depends upon the phosphorylation of a synthetic histone-based peptide substrate (PKTPKKAKKL), which has been biotinylated at its C-terminus. When this biotinylated peptide is incubated with [gamma-33P] ATP and TPK II/cdk5 under defined assay conditions, product formation is linear with respect to time and enzyme concentration. The production of [33P] phosphorylated peptide is inhibited in the presence of a known TPK II/cdk5 inhibitor but is unaffected in the presence of 1% DMSO. A signal-to-noise ratio of 16:1 was obtained in a 60-min assay with an intra-assay variability of <10% in the 96-well microtiter format. The TPK II/cdk5 SPA is very robust, sensitive and simple to perform.[1]References
- A scintillation proximity assay for studying inhibitors of human tau protein kinase II/cdk5 using a 96-well format. Evans, D.B., Rank, K.B., Sharma, S.K. J. Biochem. Biophys. Methods (2002) [Pubmed]
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