| Assay Method Information | |
| | KRAS G12C-BRAF NanoBit Assay |
| Description: | HEK293 cells were grown and maintained using DMEM medium (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin/streptomycin. Both KRAS G12C and BRAF RBD were cloned into the NanoBit vectors (BiBiT vectors system, Promega) with the orientations SmBit-KRAS G12C and BRAF RBD-LgBit, respectively, and co-transfected into HEK293 cells. Cells were then selected with 100 μg/mL Hygromycin B (10687010, Thermo Fisher) and Blasticidin (5 μg/mL) for 4 weeks to get the stable cell pool. On the day of the assay, 75 nL of compound solution was presented in a 384-well assay plate as a 16-point 3-fold dilution starting from a final concentration of 30 μM in DMSO. Then cells were seeded at 10,000 cells/25 μL/well in a 384-well plate. After 3 hours of incubation, 6 μL of volume of Nano-Glo® Live Cell Substrate (Promega) was added into each well. Monitor luminescence using ultra384 model in Envision at 20 minutes. Compounds that facilitate disruption of the KRAS G12C-BRAF RBD complex were identified as those eliciting a decrease of luminescence relative to DMSO control wells. |
| Affinity data for this assay | |
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