| Assay Method Information | |
| | Detection of Compound's Ability to Inhibit HPK1 Kinase Activity Assay |
| Description: | The specific operation is as follows: configure the enzymatic reaction system buffer (10 mM MOPS, pH 7.2, 5 mM β-glycerol-phosphate, 10 mM MgCl2, 0.8 mM EDTA, 2 mM EGTA, 0.1 mM DTT); the test compound (1 mM the compound stock solution in DMSO) was diluted with buffer to the highest concentration of 60 uM (containing 6% DMSO), and the compound with a concentration of 60 μM was initially diluted 5 times with a buffer containing 6% DMSO for a total of 8 points of gradient concentration; then dilute HPK1 kinase to 30 nM in buffer. Add 2 μl of HPK1 kinase diluent to each well of a Greiner 384-well microplate (Cat. No. 784075), and add 2 μl of buffer to the control well; add 1 μl of the diluted compound to the reaction well after brief centrifugation, and add 1 μl buffer solution containing 6% DMSO to the control well; after short centrifugation, place in a 25° C. constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd., Cat. No.: LRH-150) and incubate for 20 minutes. Add 3 μl of reaction substrate (10 μM MBP and 20 μM ATP dissolved in distilled water) to each well, briefly centrifuge and incubate in a constant temperature incubator at 25° C. for 60 min, and use ADP-Glo Kinase Assay Kit to detect the enzymatic reaction activity, ADP-Glo Kinase Assay Kit detection is carried out according to the operating instructions of the kit. Data are described using the half maximal inhibitory |
| Affinity data for this assay | |
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