| Assay Method Information | |
| | In Vitro Enzyme Activity Test |
| Description: | The IC50 values of the compounds against MST1 were determined. The protein kinase MST1 and the substrate were obtained from commercial sources.5.4 μL of protein kinase MST1 diluted to a certain concentration (final concentration was 2.5 ng/μL) and 1 μL of serially diluted drug compounds and control compound XMU-MP-1 were taken to react at room temperature for 10 minutes (final drug concentrations were 10 μM, 1 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM, respectively).6 μL of a mixture of ATP (with a final reaction concentration of 50 μM) and substrate was added to the above reaction tube to react at 37° C. for 1 hour. The reaction buffer was 40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 50 μM DTT.5 μL of the kinase mixture after the reaction was taken to a 384-well plate, and 5 μL of ADP-GLO™ reagent was added thereto. After reaction at room temperature for 40 minutes, the kinase reaction was terminated and the remaining ATP was consumed.10 μL of a kinase detection reagent was added to convert ADP into ATP, and a coupled luciferase/luciferin reaction was utilized to detect the newly synthesized ATP. |
| Affinity data for this assay | |
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