| Assay Method Information | |
| | Surface Plasmon Resonance (SPR) Assay |
| Description: | Surface Plasmon Resonance (SPR) Experiments. SPR experiments were performed on a Biacore S200 instrument at 25° C. Biotinylated Hsp90 Nuclear Binding Domain (NBD) was diluted to 40 μg/mL and immobilized on a streptavidin chip (Sensor Chip SA, GE Healthcare) at a density of 2000-2500 response units (RU) on the biosensor surface.Recombinant Hsp90 NBDs were expressed and purified as previously described (Whitesell et al., Nat Commun 10, 402 (2019)) with the following modification; Hsp90 NBD expression constructs were modified to encode a C-terminal AviTag for site-specific on-column biotinylation with a BirA biotin-ligase kit (Avidity LLC; BirA-500). Stock protein solutions in 50% glycerol were stored at −20° C. until dilution into relevant buffers. Binding experiments were done in HBS-P (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% v/v surfactant P20, GE Healthcare) with 2% DMSO at flow rate of 40 μL/min. Test compounds (dilution series) were injected with a 60 s association time and 600 s dissociation time. Resulting sensorgrams were analyzed with a fit to a 1:1 binding model, using BIA evaluation software. |
| Affinity data for this assay | |
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