| Assay Method Information | |
| | KRAS G12D LoEnz Biochemical Assay |
| Description: | Biochemical compound potencies are assessed by evaluating inhibition of SOS1I-mediated nucleotide exchange in KRAS G121D. In this assay, the SOS1-promoted exchange of fluorescently-labeled GDP (BOPIDY-GDP) is monitored by time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds solubilized in DMS0 are dispensed as concentration series into 384-well white assay plates. A preformed complex of biotin-tagged recombinant human KRAS (0.06 nM mutant G1 2D) and 0.06 nM terbium-labeled streptavidin (CisBIO) prepared in 10 uL/well assay buffer (20 mM HEPES, pH 7.5, 50 mM NaCl, 10 mMMMgCl2, 0.0100 Tween-20 and 1 mM dithiothreitol) is added and allowed to incubate for 10-minutes at room temperature. The reaction is initiated with the addition of 5 uL 300 nM recombinant human SOS1 and 300 nM BODIPY-GDP in assay buffer. After a 120-minute incubation, the fluorescence is measured with excitation at 337 nm and emission at 490 and 520 nm. The TR-FRET ratio is determined as the fluorescence at 520 nm divided by the fluorescence at 490 nm multiplied by 10,000. The results are normalized to percent inhibition based on control samples: DMSO (0% inhibition) and control compound at a concentration that inhibits completely (100% inhibition). |
| Affinity data for this assay | |
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