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

Kit Size: 200 Assays (96-well) or 500 assays (384-well)\n\nThis kit is optimized to detect the activity of Hepatitis C Virus NS3/4A protease using an\nEDANS/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. \n\nConvenient Format: Complete kit including all the assay components.\nOptimized Performance: Optimal conditions for the detection of HCV NS3/4A protease activity.\nEnhanced Value: Less expensive than the sum of individual components.\nHigh Speed: Minimal hands-on time.\n\nThe NS3/4A protease of Hepatitis C Virus (HCV) is required for the cleavage of viral\nnonstructural 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.\n\nThe 490 HCV Protease Assay Kit provides a convenient assay for high throughput\nscreening 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.\n\nKit Components:\nComponents C and D can be stored at room temperature for convenience.\nComponent A: Microtiter Plate 1x200 wells HCV NS3/4A protease substrate (250ul)\nEDANS/DABCYL FRET peptide, Ex/Em=340nm/490 nm upon cleavage\nComponent B: EDANS, fluorescence reference standard (100uM DMSO solution, 10ul)\nEx/Em=340nm/490nm\nComponent C: 2X Assay buffer (25ml)\nComponent D: Stop solution (15ml)\nComponent E: DTT (1 M, 1ml)\nComponent F: Pep4AK (150uL, 600uM), HCV NS3 protease cofactor\n\nMaterials Required (but not provided):\n96-well or 384-well microplate: Black microplate can provide better signal to noise ratio.\nFluorescence microplate reader: Capable of detecting emission at 490

Prices direct from United States Biological

Quick response times

Exclusive Biosave savings/discounts

SPECIFICATIONS

Catalog Number

H1924-82

Size

1Kit

References

1. D. L. Sali et al., Biochemistry 37, 3392-3401 (1998). 2. C. Steinkuhler et al., Biochemistry 37, 8899-8905 (1998). 3. P. Gallinari et al., J.Virol. 72, 6758-6769 (1998). 4. R. W. Hardy, J. Marcotrigiano, K. J. Blight, J. E. Majors, C. M. Rice, J.Virol. 77, 2029-2037 (2003). 5. P. Hamill and F. Jean, Biochemistry 44, 6586-6596 (2005).