CHEMBL5736969

Kinase Assay: Buffer formulation: a buffer consisted of 50 mM HEPES (pH 7.5), 0.01% BSA, 5 mM MgCl2, 0.1 mM Orthovanadate. After the buffer was formulated, an enzyme and a substrate were mixed with pre-diluted compounds of varying concentrations, and placed at room temperature for 15 minutes. The reaction was initiated by adding ATP and incubated at room temperature for 60 minutes (wherein negative and positive controls were set up). The 10 μL reaction system consisted of a 2.5 μL compound, a 5 μL mixture of enzyme and substrate, and 2.5 μL ATP. After the reaction was complete, antibodies were added to test, the detection was performed with Evnvision after incubation at room temperature for 60 minutes, and data were collected. Data analysis and simulation were performed using XLfit5 software.