| Assay Method Information | |
| | KRAS-RAF1 Binding Assay |
| Description: | This experiment is intended to investigate the inhibitory effect of compounds on the binding of KRASG12D mutant protein or KRASWt (wild type) protein to fragments of the RBD region fragment of RAF1 protein. A 500×3-fold gradient concentration stock of compound was prepared using DMSO and diluted 50-fold into a 10× stock of compound using reaction buffer. To the reaction wells of an ELISA plate (ProxiPlate-384-Plates), was first added 3 L of His-KRASG12D protein or KRASwt protein, then added 1 μL of 200 M GTP and incubated for 30 min, then added 2 μL of 10× compound or reaction buffer with 2% DMSO (positive control) and incubated for 30 min, then added 4 μL of GST-RAF1-RBD protein or reaction buffer (negative control) and incubated for 15 min, then added L of Eu-labeled anti-His antibody and XL665-labeled anti-GST antibody mixed well in advance and incubated for 60 min before reading HTRF signal by microplate reader. The inhibition ratio of compounds on KRAS protein (G12D mutant or wild type) and RAF1 binding was calculated IR (%)=(positive control signal-compound well signal)/(positive control signal-negative control signal)×100% and IC50 values were calculated by fitting compound concentrations and corresponding inhibition ratios using the Prism 8 four-parameter method. The results show that compared with KRASwt (wild type) protein, exemplary compounds of the present disclosure have higher inhibition selectivity for the binding of KRASG12D mutant protein to the RBD region fragment of RAF1 protein. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |