| Assay Method Information | |
| | Secondary Binding Analysis by Surface Plasmon Resonance |
| Description: | The His-tagged SARS-CoV-2 PLpro enzyme was initially prepared in phosphate buffer and diluted to 50 ug/mL with 10 mM sodium acetate (pH 5.5) and immobilized on a CM5 sensor chip by standard amine coupling with running buffer PBSP (10 mM phosphate, pH 7.4, 2.7 mM KCl, 137 mM NaCl, 0.05% Tween-20). The CM5 sensor chip surface was first activated by 1-ethyl-3-(3- (dimethylamino)propyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) mixture using a Biacore 8K instrument (Cytiva). SARS-CoV-2 PLpro enzyme was immobilized to flow channels 1−4 followed by ethanolamine blocking on the unoccupied surface area, and immobilization levels for all four channels were similar at 12,000 RU. Each flow channel has its own reference channel, and blank immobilization using EDC/NHS and ethanolamine was done for all reference channels. Compound solutions with a series of increasing concentrations (0.049−30 uM at 2.5-fold dilution) were applied to all active and reference channels in SPR binding buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, and 0.05% Tween-20, 0.5 mM TCEP, and 2% DMSO) at a 30 uL/min flow rate at 25 °C. The data were double referenced with a reference channel and zero concentration (2% DMSO) responses, and reference subtracted sensorgrams were fitted with 1:1 Langmuir kinetic model using a Biacore Insight evaluation software, producing two rate constants (ka and kd) (Figure S1). The equilibrium dissociation constants (KD) were determined from two rate constants (KD = kd/ ka). For steady-state affinity fittings, response units at each concentration were measured during the equilibration phase, and the KD values were determined by fitting the data to a single rectangular hyperbolic curve equation, where y is the response, ymax is the maximum response, and x is the compound concentration. |
| Affinity data for this assay | |
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