| Assay Method Information | |
| | KRAS (GDP) Biochemical Assays |
| Description: | Compounds were tested for binding to GDP-loaded wild-type KRAS (WT), KRAS-G12C, KRAS-G12D, and KRAS-G12V in a 384-well assay format using a TR-FRET probe displacement assay in buffer consisting of 50 mM Hepes (pH 7.4), 150 mM NaCl, 5 mM MgCl2 and 0.005% Tween-20.0.03 nM KRAS was used in this assay with 0.02 nM Eu-streptavidin and 12 nM Cy-5 labelled probe. Compounds were serially diluted (1:3) in DMSO. The LabCyte ECHO Acoustic dispenser was used to pre-spot the assay plates (384-well Non-Binding Surface plates, Corning, Catalog #3824) with 50 nL of compound. 5 μL of 2× enzyme concentration was added to the pre-spotted plates and incubated for 30 minutes before adding 5 μL of 2× concentration of Eu-streptavidin and Cy-5 labelled probe (10 μL final reaction volume). The plates were incubated at room temperature for 2 hours before measuring TR-FRET ratio (665 nm/615 nm) on the Envision multimode plate reader. The ratios were normalized to a positive (saturating concentration of known inhibitor) and negative (DMSO) control and plotted against the log of compound concentration. IC50 values were defined as the compound concentration that causes a 50% decrease in normalized signal and were calculated using a sigmoidal dose-response model to generate curve fits. |
| Affinity data for this assay | |
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