| Assay Method Information | |
| | KRAS WT Biochemical Assay |
| Description: | Biochemical compound potencies are assessed by evaluating inhibition of SOS1-mediated nucleotide exchange in KRAS WT. 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 DMSO are dispensed as concentration series into 384-well white assay plates. A preformed complex of 0.06 nM biotin-tagged recombinant human KRAS WT, 15 nM GDP 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 mM MgCl2, 0.01% 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. |
| Affinity data for this assay | |
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