| Assay Method Information | |
| | CDK7 Kinase Activity Assay |
| Description: | Representative compounds of the present invention were assayed in vitro for CDK7 kinase-inhibitory activities using ADP-Glo platform. In detail, CDK7 kinase-inhibitory activity was measured using recombinant purified human CDK7 kinase (ThermoFisher, PV3868) and ADP Glo kinase assays kit (Promega, V9102). CDK7 kinase was diluted with 1× kinase reaction buffer (40 mM Tris-C1, pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA and 50 [M DTT) and added to 96 well plates (final concentration of CDK7 per reaction: 50 ng). The compound was finally treated to be a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (final concentration of 90 [M) and 0.2 μg/μl of MBP (Myelin basic protein) in a total of 25 μl of reaction mass was added to 96 well plates, thereby initiating an enzymatic reaction. After 2 hours of incubation (30° C.), equivalent volume (25 μl per reaction) of ADP Glo was added and incubated (30° C.) at room temperature for 60 minutes. The kinase detection reagent (50 μl per reaction) was then added and incubated (30° C.) at room temperature for 30 minutes. The kinase activity was measured by chemiluminescence method according to ADP Glo Kanease Assay Kit instruction manual, and the CDK7-inhibitory activity of the compounds according to the present invention was calculated. The result analysis of each compound was performed using Microsoft Excel, and IC50 values were calculated by Prism software. |
| Affinity data for this assay | |
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