| Assay Method Information | |
| | RAF Inhibition Assay |
| Description: | Assay protocol: the inhibitory effects of the compounds of the present invention on the wild-type BRAF enzyme, mutant CRAF enzyme (RAF1 Y340D and Y341D) and mutant BRAF enzyme (BRAF-V600E) activity were determined according to the instructions of the TB-PMAP2K1 (PSER217/221) kit (ThermoFisher). Different RAF enzymes and substrates (FLUORESCEIN-MAP2K1) were pre-incubated with test compounds at various concentrations at room temperature for 15 min, and then the adenosine triphosphate (ATP) was added to initiate the reaction. After incubation at room temperature for 60 min, EDTA and Tb-labeled anti-pMAP2K1 [pS217/221] antibody solutions were added, and incubation was performed at room temperature for 60 min before the detection of the fluorescence values of the compounds in each group. With the vehicle group (DMSO) as the negative control and the buffer group (without RAF enzyme) as the blank control, the relative inhibitory activity percentage (i.e., the inhibition rate) of a compound at different concentrations was calculated according to the following formula:Relative inhibitory activity percentage=1−(Compound group at various concentrations−blank control)/(negative control−blank control)*100%. The relative inhibitory activity percentages of the compounds at different concentrations were plotted against the compound concentrations, the curve was fitted according to a four-parameter model, and the IC50 value was calculated according to the following formula:y=min+(max−min)/(1+(x/IC50){circumflex over ( )}(−Hillslope))wherein y is the relative inhibitory activity percentage, max and min are respectively the maximum and minimum values of the fitted curve, x is the logarithm concentration of the compound, and Hillslope is the slope of the curve. |
| Affinity data for this assay | |
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