| Assay Method Information | |
| | Biochemical Assay |
| Description: | Compounds were prepared in DMSO at 10mmol/L and serially diluted in a 96-well plate as the source plate. The intermediate plate consists of 4µL from the source plate mixed with 96µL of 1× kinase buffer (50mmol/L HEPES, pH 7.5; 10mmol/L MgCl2; 1mmol/L EGTA; 0.01% Brij-35). Of 2.5µL was transferred to a 384-well assay plate in duplicates. To the assay plate, 5µL of BRAF 7nmol/L or BRAF V600E 0.7nmol/L, 2.5µL of substrate solution containing Fluorescein-MAP2K1 0.8µmol/L and ATP (for BRAF 2µmol/L, for BRAF V600E 6µmol/L) were added and the assay plate was incubated with the compounds for 1 h at room temperature. The reaction was stopped with the addition of 10µL of detection solution (antibody 4nmol/L and EDTA 20mmol/L in antibody dilution buffer) to each well of the assay plate. |
| Affinity data for this assay | |
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