Biacore s200 instrument

Surface plasmon resonance (SPR) experiments were performed on a BIAcore™ S200 instrument (GE Healthcare) at room temperature. For all measurements, CM5 chips (Series S) and filtered PBS pH 7.4 with DMSO (5 % v/v) as flow buffer were used. Human CAIX was immobilized on the chip to 500 response units (RU) using EDC·HCl and NHS following manufacturer instructions. Serial dilutions of compounds 1 and 2 [Supplementary Figure 3] in running buffer at a flow rate of 20 μL min^-1 were used as analytes. After each cycle, the sensor surface was regenerated by a short treatment with DMSO (50 % v/v) in PBS. Sensorgrams were solvent corrected and the binding kinetics were analyzed with the BIAcore™ S200 evaluation software using a 1:1 Langmuir.

SPR binding experiments were performed on a Biacore S200 instrument (GE). Avi-K-Ras (amino acids 2-188) loaded with the non-hydrolyzable GTP analog, GppNHp, or avi-tagged Raf-RBD (amino acids 55-131) were captured on CM5 sensor chips (GE) containing amine coupled Neutravidin (12000–15000 RU). Alternatively, for some experiments, Raf-RBD was coupled directly to the CM5 sensor chips using standard amide coupling chemistry. Samples were injected at 30 µl/min and for a contact time of 60s. A 2-fold dilution series of Rigosertib was prepared (50-0.39 µM) in 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.05% Tween 20, 5 mM MgCl₂, 2.5% DMSO and injected over avi-RafRBD (350 RU). For competition experiments, 5 µM Raf-RBD was mixed with increasing concentrations of Rigosertib (0.19 – 50 µM) and injected over avi-K-Ras-GppNHp (500 RU). RafRBD binding in the presence of Rigosertib was compared to a calibrations series in which Raf-RBD 0.15 – 20 µM (2-fold dilutions) was injected over the surface. The data were processed by subtracting binding responses on the reference flow cells as well as binding responses when buffer was injected. The samples were also corrected for DMSO mismatches using a DMSO standard curve.