| Assay Method Information | |
| | Enzyme Activity Assay of CDK9 Protein Inhibition In Vitro |
| Description: | The DMSO diluted compounds and the control compounds Dinaciclib, JSH-009, BAY-1211152, AZD4573 (all purchased from MCE, China) were mixed with the detected CDK9/CyclinT1 protein (Invitrogen, U.S.), CDK9-L156F mut/CyclinT1 mutant protein (this mutation is consistent with the mutation in HepG2 KI C20-1, which was constructed as described in Example 108), and incubated at room temperature for 30 minutes; Kinase Peptide Substrate Mixture (Invitrogen, U.S.) and 4×ATP were added for mixing, and the mixing system was transferred to a 384-well white opaque plate for reaction at room temperature for 1 hour; 5 μL Development Solution (Invitrogen, U.S.) was added and reacted at room temperature for 1 hour, and finally Stop Agent (Invitrogen, U.S.) was added to terminate the reaction; the fluorescence value was read using MD SpectraMax I3X microplate (Molecular Devices, U.S.). The IC50 values of the compounds of the invention on the tested CDK9/CyclinT1 protein and CDK9 mutant protein CDK9-L156F mut/CyclinT1 were calculated by plotting using Prism 5.0 (GraphPad Software, San Diego, CA) based on the read fluorescence value, which were shown in Table 5 and Table 6. |
| Affinity data for this assay | |
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