| Assay Method Information | |
| | Surface Plasmon Resonance (SPR) SOS1 Binding AssaySurface Plasmon Resonance (SPR) SOS1 Binding Assay |
| Description: | Binding to SOS1 was measured using a SPR assay with purified recombinant human SOS1 substrate (res. 564-1049 with N-terminal Avi tag; purified and biotinylated based on Hillig, et al., Proc Natl Acad Sci USA (2019); 116(7):2551-2560). SPR measurements were performed on a Biacore 8K SPR instrument (GE Healthcare, Sweden). Assays were performed at 25° C. using Series S SA sensor chips pre-coated with streptavidin (GE Healthcare, Cat. BR100531). Biotinylated SOS1 diluted in sample buffer (20 mM Tris HCI, 150 mM NaCl, 1 mM DTT, 0.05% TWEEN 20, 1 mM MgCh, pH 8.0) was captured to one flow cell of the chip to about 3,000 resonance units (RU) using sample buffer supplemented with 5% DMSO as a running buffer. Serial dilutions of the assayed compounds in the running buffer at 100, 50 or 0.5 µM were injected for 60 s at a flow rate of 30 µL/min and association phases were recorded. Dissociation of the samples was monitored for 600 s. Data processing was performed using Biacore Insight Software (Biacore, GE Healthcare). Sensorgrams recorded on a SA flow cell without captured protein were subtracted from sensorgrams recorded on the SOS1 surface. Blank injections of running buffer were used for double referencing and solvent correction was applied to all sample sensorgrams to correct for buffer mismatches. KDS were estimated using a kinetic or steady state, where applicable, fitting model describing a reversible equilibrium with 1:1 binding between SOS1 and the compound. |
| Affinity data for this assay | |
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