| Assay Method Information | |
| | In Vitro LasB Activity Assay |
| Description: | Fluorescence intensity was measured for 60 min at 37° C. in black 384-well microtiter plates (Greiner BioOne, Kremsmünster, Austria) using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany) with an excitation wavelength of 340±nm and an emission wavelength of 415±20 nm. The assay was performed in a final volume of 50 μL of assay buffer (50 mM Tris, pH 7.2, 2.5 mM CaCl2), 0.075% Pluronic F-127, 5% DMSO) containing LasB at a final concentration of or 0.3 nM and the substrate at 150 μM. Before substrate addition, compounds were preincubated with the enzyme at 37° C. for 15 min. Experiments were performed in duplicates and repeated for at least two times. Blank controls without enzyme were performed. After blank subtraction, the slope of samples containing inhibitors (v) was divided by the slope of a simultaneously started uninhibited enzymatic reaction (v0). |
| Affinity data for this assay | |
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