| Assay Method Information | |
| | HTRF Nucleotide Exchange Assay |
| Description: | The capacity of compounds to bind KRAS G12C and KRAS wildtype was quantified using a HTRF nucleotide exchange assay. Recombinant human KRAS protein (5 nM; aa1-188 KRAS WT or KRAS containing the G12C amino acid substitution, an N-terminal 6×His-tag and leader sequence), and 2 nM Europium-labeled anti-6×His antibody were mixed in assay buffer (10 mM HEPES pH7.3, 75 mM NaCl, 5 mM MgCl2, 0.05% BSA, 0.0025% NP-40 and 100 mM KF) with various concentrations of compound in a 384-well plate and a volume of 5 μL. After a 180 minute incubation at room temperature, 5 μl of 200 nM EDA-GTP-DY647P1 diluted in assay buffer was added to the plate. Following 30-minute incubation at room temperature, time-resolved fluorescence was measured on a PerkinElmer Envision plate reader. DMSO (0.3%) and unlabeled GDP (1 μM) or equivalent tool compound were used to generate the Max and Min assay signals, respectively. Data was analysed using a four-parameter logistic model to calculate IC50 values, with at least two independent replicates were performed for each compound. |
| Affinity data for this assay | |
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