Hepatitis C Virus Protease, Fluorometric 490, BioAssay™ Kit (HCV)

Kit Size: 200 Assays (96-well) or 500 assays (384-well)||This kit is optimized to detect the activity of Hepatitis C Virus NS3/4A protease using an|EDANS/DABCYL FRET peptide substrate, with its fluorescence monitored at Ex/Em=340 nm/490nm upon proteolytic cleavage. It provides ample materials to perform 200 assays in a 96-well format and 500 assays in a 384-well plate. ||Convenient Format: Complete kit including all the assay components.|Optimized Performance: Optimal conditions for the detection of HCV NS3/4A protease activity.|Enhanced Value: Less expensive than the sum of individual components.|High Speed: Minimal hands-on time.||The NS3/4A protease of Hepatitis C Virus (HCV) is required for the cleavage of viral|nonstructural polyprotein at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. These cleavages are essential for the maturation of the viral proteins. Thus, this protease has become one of the key targets for developing anti-HCV drugs.||The 490 HCV Protease Assay Kit provides a convenient assay for high throughput|screening of HCV NS3/4A protease inhibitors and for continuous quantification of HCV NS3/4A protease activity using fluorescence resonance energy transfer (FRET) peptide. The sequence of this FRET peptide is derived from the cleavage site of NS4A/NS4B. The cysteine on the natural cleavage site is replaced with aminobutyric acid (Abu) and the scissile amide bond with an ester bond. These modifications improved kcat/Km values by more than 100 fold and enabled the detection of the activity of NS3/4A protease at subnanomolar concentrations. In the FRET peptide, the fluorescence of EDANS is quenched by DABCYL. Upon cleavage into two separate fragments by HCV NS3/4A protease at the Abu-Ala bond (see Scheme 1), the fluorescence of EDANS is recovered, and can be monitored at excitation/emission = 340 nm/490 nm.||Kit Components:|Components C and D can be stored at room temperature for convenience.|Component A: Microtiter Plate 1x200 wells HCV NS3/4A protease substrate (250ul)|EDANS/DABCYL FRET peptide, Ex/Em=340nm/490 nm upon cleavage|Component B: EDANS, fluorescence reference standard (100uM DMSO solution, 10ul)|Ex/Em=340nm/490nm|Component C: 2X Assay buffer (25ml)|Component D: Stop solution (15ml)|Component E: DTT (1 M, 1ml)|Component F: Pep4AK (150uL, 600uM), HCV NS3 protease cofactor||Materials Required (but not provided):|96-well or 384-well microplate: Black microplate can provide better signal to noise ratio.|Fluorescence microplate reader: Capable of detecting emission at 490±30nm with excitation at 340±3 nm.|HCV NS3/4A protease: HCV NS3/4A protease can be produced from E. coli1-3.||Storage and Handling:|Store all kit components at -20°C, and keep Components A and B away from light. If used frequently.