| Assay Method Information | |
| | CDK1/CyclinB Kinase Inhibitory Activity Assay |
| Description: | CDK1/CyclinB kinase inhibitory activity (IC50): 5 μl of various dilutions of test compound in 1×kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT and 0.01% Brij-35) was mixed with 10 μL of CDK1/CyclinB (Millipore, 14-450M #, final concentration 3 nM in 1×Kinase buffer) in 384 plates and incubated at room temperature for 10 min. To initiate each reaction, 10 μL of peptide solution containing fluorescently-labeled −peptide 18(5-FAM-QSPKKG-CONH2) (GL, 114202 #, final concentration 3000 nM) and ATP (final concentration 20 M) in 1×Kinase buffer was added to each of the wells containing test compound and CDK1/CyclinB mixture. The reaction is then allowed to proceed at 28° C. for 30 min and terminated by the addition of 25 μL stop buffer (100 mM HEPES pH 7.5, 50 mM EDTA, 0.2% Coating Reagent #3 (Perkin Elmer, 760050 #) and 0.015% Brij-35). |
| Affinity data for this assay | |
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