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Validation of Luminescent Assay for CDK2 Inhibitors.
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| Author:
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UPLOADED BY-ADMIN, MAULIK P. SUTHAR, RAKESH P. PATEL
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| Abstract:
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Background: Cyclin Dependent Kinases ensures the tight regulation of the cell cycle execution by mediating phosphorylation
of cellular proteins. Mis-regulation of the CDK2 activity by cellular and external factors leads to many diseases like
cancers.
Objective: Present study is focused on development, optimization and validation of CDK2 assay which is suitable for identification
potent and selective, ATP competitive inhibitors of CDK2.
Method: Luminescent Signal was developed using beetle luciferin as a substrate for determination of residual ATP leftover
after Histone H1 phosphorylation, mediated by CDK2 isoforms. Reactions were started by adding 100 µM ATP and
2.5mg/ml Histone H1, at room temperature for 20 minutes. 5µl of Kinase glo plus reagent was added and was allowed to
stabilize for 1 min, and then luminescence was recorded.
Results: Enzyme and substrate concentrations were optimized, which were 50 ng/well for CDK2/Cyclin A and 25 ng/well
for CDK2/cyclin E. Histone H1 was used as a substrate. Z’ was determined, which were 0.817 for CDK2/cyclin A and
0.772 for CDK2/cyclin E assays.
Conclusions: With Z’ factor higher than 0.7, both assays are suitable for screening of inhibitors. This can help to determine
selectivity and potency of CDK2 inhibitors.
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| Keyword:
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CDK2, Luminescence, inhibitors
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| EOI:
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| DOI:
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