| Assay Method Information | |
| | In Vitro Enzyme Activity Assay |
| Description: | Table 1: Method 1: The IC50 value was assayed by 33P isotope-labeled kinase activity assay to evaluate the inhibitory ability of a test compound on human HPK1.Enzyme buffer conditions: 50 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij35, 2 mM DTT.Preparation of the mixture of kinase and substrate: the concentration of HPK1 in the reaction solution was 0.1 nM or 8.1 nM, the concentration of ATP was 10 M or 15 M, and the concentration of MBP was 0.05 mg/mL.Assay steps: A compound was diluted 3-fold with DMSO in a dilution plate, with a final initial concentration of 10 M and 10 concentration gradient points. The compound was diluted 50-fold into the kinase reaction buffer, and the mixture was shaken on a shaker for 20 minutes. The kinase was prepared with the enzyme reaction buffer. 2 μL of kinase was added to each well of a reaction plate. 1 μL of the diluted compound in the buffer was added to each well. The plate was sealed with a sealing film, centrifuged at 1000 g for 30 seconds, and placed at room temperature for 10 minutes. The mixed solution of ATP/MBP was prepared with the enzyme reaction buffer. 2 μL of the mixed solution of ATP/MBP was added to the reaction plate. The plate was sealed with a sealing film, and centrifuged at 1000 g for 30 seconds. The mixture in the plate was reacted at room temperature for 60 minutes. 4 μL of ADP-Glo was transferred to the 384 reaction plate and centrifuged at 1000 rpm/min for 1 min. The plate was incubated at 25° C. for 40 min. 8 μL of detection solution was transferred to the 384 reaction plate, and centrifuged at 1000 rpm/min for 1 min. The plate was incubated at 25° C. for 40 min. |
| Affinity data for this assay | |
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