| Assay Method Information | |
| | Kinase Activity Assay |
| Description: | Inhibitory activities of test compounds against BRAF (Invitrogen, PV3848), cRAF (BPS Bioscience, 40008) and BRAF (V600E) (Invitrogen, 40533) were determined using ADP-GloTM Kinase Assay kit (Promega, V9102).The compounds were 3-fold serially diluted with DMSO (MP, 196055), respectively, each with 10 concentrations. 100 ml of compound diluents and 2.5 μL of BRAF or cRAF or BRAF (V600E) were added to each well of a 384-well plate (Perkin Elmer, 6007290) in duplicate. After incubating at 25° C. for 15 minutes, 2.5 μL of substrate was added to start the reaction. The plate was incubated at 25° C. for 60 minutes. The final reaction concentrations in the system were: 5 nM BRAF or 1.5 nM cRAF or 5 nM BRAF (V600E), 10 mM ATP, 200 nM MEK1, HEPES 50 mM, EGTA 1 mM, MgCl2 10 mM, and Brij35 0.01%. The concentrations of the test compounds were: 300, 100, 33.33, 11.11, 3.7, 1.23, 0.412, 0.137, 0.046, 0.015, 0 nM, and the final concentration of DMSO was 1%. Then 10 μL of ADP Glo reagent was added, and further incubated at 25° C. for 40 minutes. Then 20 μL of detection reagent was added, and further incubated at 25° C. for 40 minutes. The enzyme activity in the presence of the compounds at each concentration was then measured by an Envision microplate reader (Perkin Elmer 2104), and the inhibitory activity of the compounds at each concentration against the enzyme activity was calculated. The inhibitory activities of the compounds at different concentrations against the enzyme activity were then fitted using Graphpad 5.0 software according to the four-parameter equation, and the IC50 values were calculated. |
| Affinity data for this assay | |
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