| Assay Method Information | |
| | Biochemical Functional Assay |
| Description: | The KRAS (aa 1-169) G12C, C51S, C80L, C118S with a His-tag was expressed, purified and loaded with GDP in house. All protein and substrate solutions were prepared in assay buffer containing 25 mM HEPES pH7.5, 10 mM MgCl2, and 0.01% Triton X-100. Purified GDP-loaded KRAS (aa 1-169) G12C, C51S, C80L, C118S protein was pre-incubated with a serially diluted compound at 24° C. for 3 hrs. Purified SOS1 (aa 564-1049) protein, GTPyS (Sigma) and GST-cRaf RBD (aa 1-149) were then added to each well and incubated at 24° C. for additional 3 hrs. This addition initiates the nucleotide exchange reaction and transition of inactive GDP loaded KRAS G12C to active GTPyS KRAS G12C which binds to GST-cRaf RBD. Following the incubation, Mab Anti-6HIS-T cryptate (Cisbio) and Mab Anti GST-XL665 (Cisbio) were added and further incubated at 24° C. for 3 hrs. The binding interaction between active GTPyS KRAS G12C and GST-cRaf RBD brings the Tb and XL665 into close proximity enabling an increased FRET signal (Ex337 nm, Em665 nm/620 nm). The inhibition percentage of nucleotide exchange reaction in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 665 nm to that at 620 nm detected on a BMG PHERAstar FSX instrument. |
| Affinity data for this assay | |
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