| Assay Method Information | |
| | In Vitro Enzymatic Inhibitory Activity Assay |
| Description: | The test was performed in a U-shaped bottom 384-well plate (coming, 4512 #), and the reaction temperature was 27° C. CDK7/CyclinH was diluted in a test buffer solution (20 mM MES PH6.75, 0.01% Tween20, 50 ug/mL BSA, 6 mM MgCl2) to obtain a corresponding enzyme solution with a 2.4× concentration. CDK2/CyclinE1 was diluted in a test buffer solution (20 mM MES PH6.75, 0.01% Tween20, 50 ug/mL BSA, 6 mM MgCl2) to obtain a corresponding enzyme solution with a 2.4× concentration. CDK9/CyclinT1 was diluted in a test buffer solution (20 mM MES PH6.75, 0.01% Tween20, 50 ug/mL BSA, 10 mM MgCl2) to obtain a corresponding enzyme solution with a 2.4× concentration. CDK12/CyclinK was diluted in a test buffer solution (80 mM MES PH6.5, 0.01% Tween20, 50 ug/mL BSA, 10 mM MgCl2) to obtain a corresponding enzyme solution with a 2.4× concentration. The compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM. When in use, the compounds were diluted with DMSO into 10 concentration gradients ranging from 25 nM to 500 uM, which were respectively diluted by 8.3 times in the test buffer solution to obtain compound solutions with a 6× concentration. A polypeptide substrate and ATP were diluted in the test buffer solution to obtain a mixed solution of the polypeptide substrate and ATP with a 2.4× concentration. 2 ul of a test compound solution was mixed with 5 ul of enzyme solution. After incubating for 10 min, 5 ul of the mixed solution of the polypeptide substrate and ATP was added. After incubating at 27° C. for 180 min, 4 uL of EDTA with a 120 mM concentration was added to each sample to stop the reaction. The test buffer solution containing 20 uM of staurosporine replaced the compound solution as 100% inhibitory control, the DMSO replaced the compound solution as 0% inhibitory control, and each test at least contained 2 parallel controls. |
| Affinity data for this assay | |
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