| Assay Method Information | |
| | Fluorimetric Assay |
| Description: | done in a single pass in black 384-well plates (Matrix Technologies). The SARS-PLpro enzyme (20 nM final concentration) was prepared in an assay buffer (50 mM HEPES, pH 7.5, 0.01% Triton X-100 (v/v), 0.1 mg mL−1 BSA, and 2 mM GSH). The MERS-PLpro enzyme (400 nM final concentration) was prepared in the same assay buffer with 5 mM DTT in place of 2 mM GSH. A total of 30 μL of enzyme solutionwas dispensed into wells, and then 200 nL of 10 mM compounds (50 μM final concentrations) was added and incubated for 5 min. Enzyme reactions were initiated with 10 μL of substrate Z-Arg-Leu-Arg-Gly-Gly-AMC (Bachem Bioscience; 50 μM and 75 μM for SARS- and MERS-PLpro, respectively) dissolved in the assay buffer and incubated for 6 min, followed by adding 10 μL of 10% SDS (w/v) as a stop solution. Fluorescence intensity was monitored at 360 nm (excitation) and 450 nm (emission). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |