An Edans-Dabcyl FRET platform was established, following a published protocol13. Briefly, a consensus cleavage sequence recognised by SARS-CoV-2 3CLpro was synthesised by Genomics, Taiwan, with Dabcyl at the N-terminus and Edans at the C-terminus, Dabcyl-TSAVLQ↓SGFRKME-Edans. In protease activity assays, 0.25 µM protease was incubated with 1.25 µM peptide substrate for three hours. Assays were conducted in triplicate in Eppendorf® black 96-well microplates (MA, USA) using an assay buffer containing 12 mM Tris-HCl (pH 7.5), 120 mM NaCl, 0.1 mM EDTA and 1 mM dithiothreitol (DTT), in a final volume of 100 µL. The fluorescence signal at 538 nm, at a bandwidth of 15 nm, emitted from the cleaved IQF peptide substrate after excitation at 355 nm, at a bandwidth of 10 nm, was recorded by a SPARK® multimode microplate reader (TECAN, Switzerland). The relative fluorescence units (RFU) at a gain of 131 were calculated using Spark® Control Magellan™ v2.2 software.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.