| Assay Method Information | |
| | SARS-CoV-2, SARS-CoV-1, MERS, 0C43 and 229E Mpro Biochemical Protease Activity Assay |
| Description: | SARS-CoV-2 Mpro biochemical assays were performed in 8 μL in 384-well Proxi-Plus plate (PerkinElmer, Waltham, MA USA) at ambient temperature in the optimized assay buffer (50 mM Hepes, pH 7.1, 800 mM sodium citrate, 1 mM ethylenediaminetetraacetic acid, 1 mM TCEP ((tris(2-carboxyethyl)phosphine)), 0.005% BSA (bovine serum albumin)) with 1 nM of recombinant Mpro and 5 μM substrate. After 10 minutes of preincubation of inhibitor with Mpro in assay buffer, reactions were initiated by the addition of a FRET-compatible peptide substrate (TAMRA)-dPEG2-S-A-V-L-Q-S-G-dPEG2-K(QSY7)-amide. Fluorescence was measured at 20 minutes using a 531/579 excitation/emission filters on EnVision (PerkinElmer, Waltham, MA USA). Data were normalized to DMSO minus no enzyme controls. IC50 and Ki of inhibitor was analyzed using Dotmatics software on 11 points of inhibitor with varies concentration. Each inhibitor was tested at least twice at different dates. 2 nM Mpro and 5 μM substrate for SARS-CoV-1, 5 nM Mpro and 5 μM for MERS, 1 nM of Mpro and 2 μM substrate for 0C43, 3 nM Mpro and 2 μM substrate for 229E were used in respective Mpro biochemical protease activity assays. |
| Affinity data for this assay | |
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