| Assay Method Information | |
| | 3CLpro enzyme assay |
| Description: | The 3CLpro enzyme assay was developed in 384-well black, medium binding microplates (Greiner Bio-One, Monroe, NC, USA) with a total volume of 20 μL and then miniaturized to 1536-well format. In 384-well plate format, 10 μL enzyme in reaction buffer was added into each well, followed by the addition of 10 μL substrate. Fluorescent intensity was measured at different time points on a PHERAstar FSX plate reader (BMG Labtech, Cary, NC, USA) with Ex=340 nm/Em=460 nm after the addition of substrate. The experiment was conducted at both room temperature (RT) and 37 °C.Steady-state kinetic parameters were evaluated using 50 nM 3CLpro and different concentrations of substrate. In brief, 10 μL/well enzyme was added into 384-well plate. The reaction was then initialized by adding the substrate solutions at different concentrations. The substrate stock solution was serially diluted 1:2 to obtain seven concentrations. The final concentrations used in this test were 160, 80, 40, 20, 10, 5, and 2.5 μM. The fluorescent intensity was measured at 5, 10, 15, and 30 min. |
| Affinity data for this assay | |
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