The SensoLyte® 520 HIV Assay Kit uses a new FRET peptide substrate that incorporates HiLyte™ Fluor 488 (fluorophore) and QXL™ 520 (quencher) for continuous measurement of enzyme activities. In the intact FRET peptide, the fluorescence of HiLyte™ Fluor 488 is quenched by QXL™ 520. Upon cleavage of the FRET peptide by HIV protease, the fluorescence of HiLyte™ Fluor 488 is recovered and can be continuously monitored at excitation/emission = 490 nm/520 nm. With superior fluorescence quantum yield and longer emission wavelength, this HiLyte™ Fluor 488/QXL™ 520-based FRET peptide shows less interference from autofluorescence of test compounds and cellular components, thus providing better assay sensitivity. The assays are performed in a convenient 96-well or 384-well microplate format.
Component A: HIV-1 protease FRET substrate: 120 µL
Component B: HiLyte Fluor™488, fluorescence reference standard Ex/Em=490 nm/520 nm: 100 µM, 5 µL
Component C: Pepstatin A, a characterized HIV-1 protease inhibitor: 27.4 µg powder
Component D: 2X Assay buffer 20 mL
Component E: Stop solution: 10 mL
Component F: DMSO: 50 µL
Component G: DTT: 1 M, 150 µL
Storage & stability
Store all components at -20°C. Components E and F can be stored at room temperature for convenience. Protect Components A and B from light.