CHEMBL5737161

Kinase Inhibitory Activity Assay: 1.2.2 Preparation of kinase solution: Kinase solutions having the concentrations required for each reaction system were prepared from 100 ng/μl kinase stock solution and the 1× kinase reaction buffer. 1.2.3 Preparation of the test compound solution and control LY2835219 solution: (1) Preparation of control LY2835219 solution a. 1 μl of 10 mM standard substance stock solution was taken and added to 9 μl of kinase reaction buffer I and mixed well; then 90 μl of kinase reaction buffer I was added and mixed well; then 100 μl of kinase reaction buffer I was added and mixed well. The final concentration was 50 μM. b. 40 μl of kinase reaction buffer II was added into B2 to B10 of a 96-well plate, and 50 μl of the above solution was added into B1; c. 10 μl of solution was taken from well B1, added into B2, and mixed well; then 10 μl of the resulting solution was taken and added into B3, and dilution was carried out in sequence to B9, so as to obtain control solutions that are diluted by 5-fold sequentially. (2) Preparation of test compound solution: a. Test compound solutions at a certain concentration were taken, respectively, diluted with kinase reaction buffer I to a final concentration of 50 μM compound solution; b. 40 μl of kinase reaction buffer II was added into H2 to H10 of the 96-well plate; and 50 μl of the above solution was added into H1; c. 10 μl of solution was taken from well H1, added into H2, and mixed well; then 10 μl of the resulting solution was taken and added into H3, and dilution was carried out in sequence to H9, so as to obtain test compound solutions that are diluted by 5-fold sequentially. 1.2.4 Preparation of a mixed solution of reaction substrate and ATP: a. Preparation of ATP solution: 200 μl of 0.1 mM ATP solution: 2 μl of 10 mM ATP was added to 198 μl of kinase reaction buffer I; 300 μl of 50 μM ATP solution: 150 μl of kinase reaction buffer I was added to 150 μl of the above 0.1 mM ATP solution; b. Preparation of 300 μl of reaction substrate solution: 150 μl 1 μg/μl reaction substrate stock solution was added to 150 μl kinase reaction buffer I, and mixed well; c. The above a/b solutions were mixed to obtain mixed solutions, respectively.