| Assay Method Information | |
| | PLpro Primary Assay |
| Description: | The PLpro primary assay, which measures protease activity with the short peptide substrate Z-RLRGG-AMC (Bachem), was performed in black flat-bottom 384-well plates containing a final reaction volume of 50 uL. The assays were assembled at room temperature as follows: 40 uL of 50 nM PLpro in buffer B (50 mM HEPES, pH 7.5, 0.1 mg/mL BSA, 0.01% Triton-X 100, and 5 mM DTT) was dispensed into wells containing 0.1−1 uL of inhibitor in DMSO or appropriate controls. The enzyme was incubated with inhibitor for 10 min prior to substrate addition. Reactions were initiated with 10 uL of 62.5 uM RLRGG-AMC in buffer B. Plates were shaken vigorously for 30 s, and fluorescence from the release of AMC from peptide was monitored continuously for 15 min on a Tecan Infinite M200 Pro plate reader (λexcitation = 360 nm; λemission = 460 nm). Slopes from the linear portions of each progress curve were recorded and normalized to plate-based controls. Positive control wells, representing 100% inhibition, included 10 uM GRL0617; negative control wells, representing 0% inhibition, included the vehicle. The selectivity of the most potent inhibitors was tested against the human deubiquitinating enzymes USP7 and USP14 (Boston Biochem). Assay conditions were similar to the PLpro primary assay, with the following substitutions: USP7 assays contained 4 nM USP7 and 0.5 uM Ub-AMC (Boston Biochem); USP14 assays contained 1.7 uM USP14, 4 uM Ub-AMC, and the addition of 5% glycerol to buffer B. PLpro activity with ISG15-AMC and Ub-AMC were assayed in a manner similar to the PLpro primary assay. PLpro and substrate concentrations were modified as follows: 80 nM PLpro was assayed with 0.5 uM Ub-AMC, and 4 nM PLpro was assayed with 0.5 uM ISG15-AMC. |
| Affinity data for this assay | |
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