| Assay Method Information | |
| | SOS1/KRAS-G12D Binding Inhibition Assay |
| Description: | SOS1 binding affinity of candidate compounds was measured by monitoring the interaction of SOS1 with KRAS-G12D in the presence of the test compound. Briefly, 5 nM GST tagged SOS1 proteins (final concentration) were pre-incubated with 50 nM 6*His tagged KRAS-G12D proteins (final concentration) in an assay buffer containing 10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.1% Tween-20, and 0.05% BSA for 30 minutes. The test compounds in 2% DMSO (final concentration) at various concentrations were then added to the reaction mixture and incubated for another 30 minutes at 4 C. 5 ug/ml GSH AlphaScreen donor beads (PerkinElmer, 6765300) and 5 ug/ml nickel chelate (Ni-NTA) AlphaScreen acceptor beads (PerkinElmer, 6760619C) (final concentrations) were then added to the mixture. After an incubation of 2 hours at 4 C., the fluorescent signals were obtained on the EnVision 2105 Multilabel Plate Reader (PerkinElmer). |
| Affinity data for this assay | |
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