| Assay Method Information | |
| | Kinase Activity Assay |
| Description: | The inhibitory effect of the compound on the kinase CDK4/cyclin D3 was tested by the Caliper Mobility Shift Assay method. The final test concentration of the compound was 10 concentrations, which started from 1p M and were obtained by three-fold dilution. 5 μL of 5-fold final concentration compound and 10 μL of CDK4/Cyclin D3 kinase solution with final concentration of 10 nM were added to the 384-well reaction plate respectively, and pre incubated at room temperature for 10 minutes (the negative control well contained 10 μL kinase buffer and 5 μL 5% DMSO; the positive control well contained 10 μL kinase solution and 5 μL 5% DMSO). The mixture of 10 μL ATP with a final concentration of 250 μM and the corresponding substrate peptide was added to initiate the reaction, and the mixture was reacted at room temperature for 150 minutes. 30 μL of stop test solution containing EDTA was added to stop the kinase reaction. Conversion rate was read by Caliper EZ Reader. Conversion inhibition rate %=(average conversion rate of positive control %−sample conversion rate %)/(average conversion rate of positive control %−average conversion rate of negative control %). Wherein: the negative control well represents conversion rate reading of wells without enzyme activity; the positive control well represents conversion rate reading of wells without compound inhibition. The log value of concentration was taken as the X-axis and the percentage inhibition rate was the Y-axis. The dose-effect curve was fitted by log(inhibitor) vs. response—Variable slope of the analysis software GraphPad Prism 5, and the IC50 value of each compound on the enzyme activity was obtained. Calculation formula: Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log IC50−X)*HillSlope)). |
| Affinity data for this assay | |
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