The SensoLyte® 520 HCV Assay Kit uses a new FRET peptide substrate that incorporates 5-FAM (fluorophore) and QXL™ 520 (quencher) for a continuous measurement of enzyme activities. In the intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL™ 520. Upon cleavage of the FRET peptide by HCV NS3/4a protease, the fluorescence of 5-FAM is recovered and can be continuously monitored at excitation/emission = 490 nm/520 nm. With superior fluorescence quantum yield and longer emission wavelength, this 5-FAM/QXL™ 520-based FRET peptide shows less interference from autofluorescence of test compounds and cellular components. This assay is ten times more sensitive than an EDANS/DABCYL based assay and can detect 0.1 pmole of HCV NS3/4a protease. The assays are performed in a convenient 96-well or 384-well microplate format.
Component A: HCV NS3/4A protease substrate Ex/Em=490 nm/520 nm upon cleavage: 120 µL
Component B: 5-FAM, fluorescence reference standard Ex/Em=490 nm/520 nm upon cleavage: 100 µM, 5 µL
Component C: 2X Assay buffe:r 10 mL
Component D: Stop solution: 10 mL
Component E DTT: 1 M, 0.5 mL
Component F: Pep4AK: 50 µL, 600 µM
Storage & stability
Store all kit components at -20°C. Protect Components A and B from light. Components C and D can be stored at room temperature for convenience.