| Assay Method Information | |
| | BRAFV600E Enzyme Activity Test |
| Description: | BRAFV600E (ABCAM, ab204154) and MEK1K97R (USBio, M2865-06J) proteins were diluted at an appropriate dilution factor using 1× Assay buffer (PH=7.4 Tris-HCl buffer, supplemented with 10 mM MgCl2) such that the final concentration of BRAFV600E was 10 ng/μL and the final concentration of substrate MEK1K97R was 1 μM. 1 μL of BRAFV600E, 1 μL of compound serial dilution (final concentration starting from 2 μM, 5-fold dilution, 8 concentrations), and 6 μL of Assay buffer were pipetted to a 384-well reaction plate with a capacity of 20 μL. The plate was pre-incubated in a constant temperature incubator at 37° C. for 30 min. 1 μL of 200 μM ATP and 1 μM MEK1K97R were added to the reaction wells corresponding to the compound incubation, mixed evenly by vortexing, and pre-incubation was performed in a constant temperature incubator at 37° C. for 60 min for enzymatic reaction. After the reaction was completed, 5 μL of the above reaction product was pipetted to another 384-well plate, and 5 μL of formulated ADP-Glo· Reagent (Promega, V9101) was added and mixed evenly by pipetting, and then the plate was placed at room temperature for 40 min. 10 μL Kinase Detection Reagent was added to the 384-well plate and incubated at room temperature for 40 min. Finally, a microplate reader (BMG LRBTECH) was used and Luminescence module was selected to detect the LUM fluorescence value of each well. |
| Affinity data for this assay | |
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