| Assay Method Information | |
| | Kinase Activity Assay |
| Description: | Assay of Kinase Activity The inhibitory effect of compounds against the kinase CDK4/cyclin D3 was detected by Caliper Mobility Shift Assay method. The final concentration of the compounds to be tested was set at 10 concentrations starting from 1 uM by 3-fold serial dilution. 5 μL of compounds at 5-fold final concentration and 10 μL of CDK4/cyclin D3 kinase solution at a final concentration of 10 nM was added to a 384-well reaction plate, respectively. The plate was pre-incubated for 10 min at room temperature (with negative control wells containing 10 uL of kinase buffer and 5 uL of 5% DMSO; positive control wells containing 10 μL of kinase solution and 5 uL of 5% DMSO). The reaction was initiated by adding 10 μL of ATP at a final concentration of 250 uM and the corresponding substrate peptide mixture at room temperature for 150 minutes. 30 uL of stop detection solution containing EDTA was added to stop the kinase reaction. Caliper EZ Reader was used to read the conversion rate. Conversion inhibition rate %=(average conversion rate of positive control %−conversion rate of sample %)/(average conversion rate of positive control %−average conversion rate of negative control %). wherein: negative control wells represent the conversion rate readings of wells without enzymatic activity; positive control wells represent conversion readings for wells with no compound inhibition. |
| Affinity data for this assay | |
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