| Assay Method Information | |
| | Biological Assay |
| Description: | CDK2 IC50 Materials and solutions:HEPES (Sigma, H3375).Sodium orthovanadate (Sigma, 450243).DTT (Sigma).MgCl2 (Sigma, M1028).PEG-20000 (Sigma, 95172).ADP-Glo (Promega, V9102, includes ATP).Human CDK2/cycE1 (ProQinase, 0050-0055-1).Histone H1 (Merck Millipore, 14-155).Assay Procedure:A reaction mixture (6 μL) containing the following components was prepared: 60 mM HEPES (60 mM, pH 7.5), sodium orthovanadate (3 μM), PEG-20000 (50 μg/mL), DTT (1.2 mM), MgCl2 (3 mM), purified human CDK2/cycE1 (4 μg/mL), histone H1 (50 μg/mL), ATP (20 μM) and test compound at the appropriate concentration such that the final concentration of DMSO was 1% w/w. The reaction mixture was incubated at 30° C. for 75 min and then stopped by the addition of ADP-Glo Reagent (6 μL). The reaction was incubated at 25° C. for 1 h to deplete the remaining ATP. Subsequently, Kinase Detection Reagent (12 μL) was added and the reaction was allowed to proceed for 1 h at 25° C. before analysis by luminescence measurement using an Envision Plate Reader (Perkin Elmer). |
| Affinity data for this assay | |
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