| Assay Method Information | |
| | Activity Inhibition Assay on CDK Family Kinases |
| Description: | The inhibiting effects on the kinase activity of CDK1/CycB (Carna bioscience, #04-102), CDK2/CycA (Carna bioscience, #04-103) and CDK9/CycT (Carna bioscience, #04-110) were determined by using LANCE Ultra method.The kinase activity test was performed using a system (10 μL) containing the following components: substrate diluent prepared by mixing CDK kinase diluent, Ulight-Myelic basic protein (Perkin Elmer, #TRF-0109, hereinafter referred to as U-MBP) and ATP (Thermo scientific, #PV3227); and the compounds (i.e., analyte) prepared in the examples of the present disclosure. The assay for each kinase includes three test groups, i.e., a background group (Blank), a non-inhibition group (PC), and compound testing group (Test). The components included in each test group were as follows:Kinase Substrate CompoundBlank 1.33 reaction buffer Substrate diluent 2% DMSO onlyPC Kinase diluent Substrate dilution 2% DMSO onlyTest Kinase diluent Substrate dilution Compound diluentWorking concentrations of the respective components of the Test groups in different kinase reactions were as follows.Compounds: 10 mM of the compound to be tested was dissolved at room temperature and diluted with DMSO to gradient concentrations, followed by diluting with deionized water to prepare a 4 working solution of the compound, and the content of DMSO was 2%. The highest concentration of compound used in the CDK1 and CDK2 assays was 10 μM, and that in CDK9 assay was 1 μM.1.33 reaction buffer: the components include 26.7 mM of MOPS, 6.67 mM of MgCl2, and 0.0133% Tween-20, which were stored in a 4 C. refrigerator and protected from light after preparation. Freshly prepared DTT was added to a final concentration of 5.33 mM before use. |
| Affinity data for this assay | |
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