| Assay Method Information | |
| | Measurement of SARS-CoV 3CLpro activity using FRET |
| Description: | To establish a high-throughput screening method, we designed a FRET peptide, Abz-SAVLQSGFRK-Dnp, as 3CLpro substrate. The FRET peptide was synthesized by Open Biosystems (Huntsville, AL). Inhibition of enzyme activity was assayed by preincubating the enzyme (20 nM) with the test compound (10 lM) in buffer B for 15 min at 25 C in 96-well plates, then adding 30 lM FRET peptide, incubating for another 15 min at 25 C, and measuring the cleaved product on a 96-well plate spectrophotometer (Safire, Tecan, Salzburg, Austria) using an excitation wavelength of 320 nm and an emission wavelength of 425 nm. Control reactions were carried out using the same reaction mixture without the test compound (no inhibition) or without the enzyme (blank). This method can also be used in an automatic robotic system for a high-throughput screen platform. |
| Affinity data for this assay | |
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