| Assay Method Information | |
| | Inhibition of Kinase Assay |
| Description: | Reagents and MaterialsRet wt (Carna, Cat. No. 08-159-10 ug), RET (V804M), Active (Signalchem, Cat. No. R02-12GG), HTRF KinEASE-TK kit (Cisbio, Cat. No. 62TK0PEC), CEP-32496 (MCE, Cat. No. HY-15200), ATP (Sigma, Cat. No. A7699), DMSO (Sigma, Cat. No. D8418-1L), DTT (Sigma, Cat. No. D0632), MgCl2 (Sigma, Cat. No. M1028), 384-well plate (Labcyte, Cat. No. P-05525-BC).Specific Assay Procedure:Formulation of compound: The test compounds were dissolved in DMSO to obtain 10 mM stock solutions, which were then serially diluted 3-fold in DMSO to obtain 10 concentrations. At the time of addition, the compound was further diluted 10-fold with buffer.Ret Wt and RET V804M Kinase Detection:In 5× kinase buffer A, Ret wt or RET V804M kinase was mixed with different concentrations of compounds prepared by pre-dilution for 10 minutes. Each concentration was tested in duplicate. The corresponding substrate and ATP were added, and reaction was performed at room temperature for 20 minutes (negative and positive controls were provided: the negative control was a blank control, and the positive control was CEP-32496). After completion of the reaction, detection reagents (reagents in the HTRF KinEASE-TK kit) were added. After incubating for 30 minutes at room temperature, enzyme activity in the presence of various concentrations of the compounds of the present disclosure was determined by Envision microplate reader, and inhibitory activity of different concentrations of the compounds on enzyme activity was calculated. The inhibitory activity of different concentrations of the compounds on enzyme activity was fitted by Graphpad 5.0 software,and the IC50 value was calculated. |
| Affinity data for this assay | |
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