| Assay Method Information | |
| | Surface Plasmon Resonance (SPR) Biosensor Assay |
| Description: | The SPR experiments were performed using a Biacore S200 instrument and CM5 biosensor chips (Cytiva, Uppsala, Sweden) at 25 C. Streptavidin (Sigma) was immobilized by amine coupling. The CM5 chip surface was activated by an injection of a 1:1 mixture of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (Cytiva, Uppsala, Sweden) or 7 min at a flow rate 10 uL/min. Streptavidin (Sigma) was diluted to 250 ug/mL in sodium acetate buffer (pH 5.0) and injected over the activated surface at a flow rate 2 uL/min for 10 min. The surface was then deactivated by the injection of 1 M ethanolamine (Cytiva, Uppsala, Sweden) for 7 min. Subsequently, the biosensor chip was conditioned with four pulse injections of 1 M NaCl/50 mM NaOH solution. Mpro was diluted to 100 ug/mL in 1.02 running buffer (50 mM TrisHCl, pH 7.5, 0.05% Tween-20) and injected at the flow rate of 2 uL/min, reaching a typical immobilization level of 8000-9000 RU. After immobilization, compounds were injected over the surface using a 10-point concentration series, at a flow rate 30 uL/min in 50 mM TrisHCl, pH 7.5, 0.05% Tween-20. An association phase was monitored for 60 s and a dissociation phase for 120 s. Sensorgrams were double-referenced by subtracting the signals from a reference surface and the signal from one blank injection. A solvent correction accounting for 2% DMSO was performed. |
| Affinity data for this assay | |
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