| Assay Method Information | |
| | Fluorescence Resonance Energy Transfer Assay |
| Description: | In this experiment, the activity of SARS-COV-2 3CLpro/Mpro protease was detected by fluorescence resonance energy transfer, and the half inhibitory concentration IC50 of the compound on SARS-COV-2 3CLpro/Mpro protease was obtained.1. Experimental MaterialsNovel coronavirus Mpro/3CLpro inhibitor screening kit (P0315M), purchased from Beyotime Biotechnology:2. Experimental Method1) The enzyme solution was prepared with reaction buffer, 49.5 μL enzyme solution was added into each well, and 49.5 μL reaction buffer was added into Min-well.2) The IC50 of the compound was detected, and the final concentration of the test was starting from 10 μM, 3-fold dilution, 10 concentrations, and each concentration was set up for multiple well testing. The compound to be tested was diluted into a solution of 200-fold final concentration, and 250 nL gradient diluted compound to be tested was added into the 384-well reaction plate by D300e (TECAN) ultramicro sampler, 250 nL 100% DMSO was transferred into both Max-well and Min-well.3) The reaction plate was incubated on ice for 10 minutes.4) 250 nL substrate solution was added into each well by D300e (TECAN).5) The reaction plate was centrifuged at 1000 rpm for 1 min, and the fluorescence signal was read continuously for 30 minutes by Envision microplate reader (PerkinElmer).6) GraphPad Prism 8 software was used to analyze data and calculate the IC 50 of the compound. |
| Affinity data for this assay | |
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