| Assay Method Information | |
| | CDK Kinase Assays |
| Description: | To demonstrate that the compounds exhibit affinity for CDK kinases (CDK2/CycA2, CDK4/CycD3, CDK6/cycD3), CDK kinase assays were performed.Reaction buffers were prepared as follows: kinase base buffer for CDK2,6 (50 mM HEPES, pH 7.5; 0.0015% Brij-35; 10 mM MgCl2; 2 mM DTT); Kinase base buffer for CDK4 (20 mM HEPES, pH 7.5; 0.01% Triton X-100; 10 mM MgCl2; 2 mM DTT); Stop buffer (100 mM HEPES, pH 7.5; 0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTA).Enzyme Reaction Protocol:1) Dilute the compound to 50X of the final desired highest concentration in reaction by 100% DMSO. Transfer 100 μL of this compound dilution to a well in a 96-well plate. Then, serially dilute the compound by transferring 30 μL to 60 μL of 100% DMSO in the next well and so forth for a total of 10 concentrations. Add 100 μL of 100% DMSO to two empty wells for no compound control and no enzyme control in the same 96-well plate. Mark the plate as source plate.2) Prepare intermediate plate by transferring 10 μL of compound from source plate to a new 96-well plate containing 90 μL of kinase buffer as the intermediate plate.3) Transfer 5 μL of compound from the 96-well intermediate plate to a 384-well plate in duplicates.4) Add 10 μL of 2.5× enzyme solution to each well of the 384-well assay plate.5) Incubate at room temperature for 10 min.6) Add 10 μL of 2.5× substrate solution prepared by adding FAM-labeled peptide and ATP in the kinase base buffer. Reaction concentrations for enzymes and substrates as following table (Table 13):TABLE 13Enzyme ATP PeptideEnzyme (nM) (μM) Peptide concentration(μM)CDK2 10 30 P18 3CDK4 10 280 P8 3CDK6 15 800 P8 37) Incubate at 28° C. for specified period of time.8) Add 25 μL of stop buffer to stop reaction.9) Collect data on Caliper. Then convert conversion values to inhibition values.Percent inhibition=(max−conversion)/(max−min)*100. max stands for DMSO control and min stands for low control herein.10) Curve fitting using percent inhibition in XLFit excel add-in version 4.3.1 to obtain IC50 values. Equation used is: Y=Bottom+(Top-Bottom)/(1+(IC50/X){circumflex over ( )}HillSlope). Wherein, Y is inhibition percentage (%); X is concentration of the test compound. |
| Affinity data for this assay | |
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