A scintillation proximity assay for studying inhibitors of human tau protein kinase II/cdk5 using a 96-well format
| Autor: | Kenneth B. Rank, David B. Evans, Satish K. Sharma |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Biophysics
Peptide In Vitro Techniques Protein Serine-Threonine Kinases Biochemistry Substrate Specificity Histones GSK-3 Alzheimer Disease Animals Humans Amino Acid Sequence Phosphorylation chemistry.chemical_classification Chemistry Cyclin-dependent kinase 5 Cyclin-Dependent Kinase 5 Molecular biology Phosphorylated Peptide Cyclin-Dependent Kinases Enzyme Scintillation proximity assay nervous system Biotinylation Scintillation Counting Cattle Oligopeptides Phosphorus Radioisotopes |
| Zdroj: | Journal of biochemical and biophysical methods. 50(2-3) |
| ISSN: | 0165-022X |
| Popis: | 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 of10% in the 96-well microtiter format. The TPK II/cdk5 SPA is very robust, sensitive and simple to perform. |
| Databáze: | OpenAIRE |
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