| Assay Method Information | |
| | Enzymatic Assay |
| Description: | The PLpro primary assay using PLpro as prepared above was conducted as described below and provided the data described in Table 7 in the column: Enzymatic assay IC50 . The PLpro primary assay, which measures protease activity with the short peptide substrate Z-RLRGG-AMC (SEQ ID NO. 4) (Bachem), was performed in black, flat-bottom 384-well plates containing a final reaction volume of 50 μL. The assays were assembled at room temperature as follows: 40 μL 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 μL 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 μL of 62.5 μM RLRGG-AMC (SEQ ID NO. 4) 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 μM GRL0617; negative control wells, representing 0% inhibition, included vehicle. |
| Affinity data for this assay | |
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