CoV Protease Substrate-2 IF670,100tests

A fluorescence resonance energy transfer (FRET)-based peptide substrate from STEMCELL Technologies, designed for the detection of protease activity of coronaviruses. The coronavirus (CoV) main protease, also called 3CLpro, regulates the CoV replication complex and is a potential target for therapeutic applications. This substrate is cleaved by 3CLpro CoV proteases and consists of a 12-amino-acid sequence (VNSTLQSGLRKM) bearing the Tide Quencher™ 5 (TQ5; quencher) and iFluor™ 670 (IF670; donor) fragments at the N- and C-termini, respectively. When intact, the favorable energetic overlap of the IF670 excited state and TQ5 absorption leads to efficient intramolecular fluorescence quenching; cleavage by CoV proteases restores fluorescence of the iFluor™ 670 moiety, which is directly correlated to CoV protease activity.

Key optical and quality specifications include an excitation wavelength of 656 nm, an emission wavelength of 670 nm, a cutoff of 660 nm, an extinction coefficient of 250,000 cm⁻¹M⁻¹, and a purity of ≥ 95%. The product is lyophilized from a dimethylformamide (DMF) solution using vacuum drying. It is stable at -20°C and should be protected from prolonged exposure to light. The vial should be centrifuged before opening and reconstituted in DMSO without vortexing; single-use aliquots should be prepared and stored at -20°C, avoiding repeated freeze-thaw cycles.

To prepare a working solution, dilute the substrate 1 in 200 in 20 mM Tris buffer (pH 7.5) or buffer of choice, and use 50 μL of substrate solution per assay in a 96-well plate. For research use only; not intended for human or animal diagnostic or therapeutic uses.