| Assay Method Information | |
| | GTP-KRAS and cRAF Interaction Assay |
| Description: | GppNp-loaded HIS-KRAS(G12D, aa 1-169) was pre-incubated with a compound in the presence of 200 uM GTP in a 384-well plate (Greiner) for 15 min, then cRAF RBD(GST tag, aa 50-132, CreativeBioMart), MAb Anti GST-d2 (Cisbio) and MAb Anti 6HIS-Tb cryptate Gold (Cisbio) were added to the assay wells (Final concentration: 2.0 nM GppNp-loaded HIS-KRAS(G12D), 100 uM GTP, 35 nM cRAF RBD, 1 ug/mL MAb Anti GST-d2, 52.5 ng/mL MAb Anti 6HIS-Tb cryptate Gold) and incubated for 2 hours at 25 C. Wells containing same percent of DMSO served as vehicle control, and wells without KRAS served as low control. HTRF signals were read on Tecan Spark multimode microplate reader and HTRF ratios were calculated under manufacturer's instructions. The percent of activation of compounds treated wells were normalized between vehicle control and low control. Then the data were analyzed using a 4-parameter logistic model to calculate IC50 values. |
| Affinity data for this assay | |
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