| Assay Method Information | |
| | Inhibition Assay |
| Description: | The PLpro enzyme was purified as described above and prepared in assay buffer (50 mM HEPES, pH 7.5, 0.01% Triton X-100 (v/v), 0.1 mg mL-1 BSA, and 2 mM DTT). IC50 values were measured in triplicate. A series of increasing concentrations (0-100 μM final concentration at 3-fold serial dilution) in 100% DMSO were prepared in a 384-well plate. 7 μL of 225 nM (3X) enzyme solution was distributed into wells, and 7 μL of varying concentration of 3X compounds were added and incubated for 10 min and 60 min for non-covalent inhibitors and covalent inhibitors, respectively. The enzyme reaction was initiated by adding 7 μL of the 75 μM (3X) substrate, and its activity was continuously monitored for at least 10 min. |
| Affinity data for this assay | |
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