| Assay Method Information | |
| | Surface Plasmon Resonance |
| Description: | Table B1: KRas G12V: Compounds were pre-dispensed via Echo liquid handling (acoustic, touch-free) to generate assay ready plates (ARP). More precisely, a 10-point concentration response curve for each compound was prepared with appropriate top concentration (1 uM, 10 uM, 100 uM). Biotinylated KRas G12V (aa 1-169) loaded with GDP nucleotide and Streptavidin-Europium (SA-EU, Columbia Biosciences) were preincubated in assay buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% Brij-35, 0.3 mg/mL BSA) on ice for 30 min. Separately SOS1 (aa 546-1049) and EDA-GTP-DY647P1 (Jena Bioscience) were preincubated in assay buffer on ice for 30 min. After 30 minutes of incubation for the assay components, 5 uL buffer and 5 uL of KRas G12V-SA-Eu were added to each well of the ARP. After a further 30 min, 5 uL of SOS1-EDA-GTP-D467P1 was added to start the nucleotide exchange reaction. Final concentration in the assay were 5 nM KRas G12V, 50 nM SOS1 (aa 564-1049). The reaction was allowed to proceed for 60-90 minutes after which the plate was read on a plate reader. After normalization of the raw data, the data was fit to a four-parameter logistic curve. |
| Affinity data for this assay | |
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