This kit is optimized to detect West Nile Virus protease activity. It provides enough reagents to perform 500 assays in 96-well format.
High Speed: The entire process only takes about half an hour.
West Nile virus (WNV) is from the family Flaviviridae3 found in both tropical andtemperate regions. The main route of human infection is through the bite of an infected mosquito Infection causes severe neurological disease and fatalities in both human and animal hosts. There is currently no effective vaccine or antiviral drug to protect against WNV infection.4WNV contains a single-stranded, positive-sense RNA genome, which encodes threestructural proteins (capsid (C), membrane (M), envelope (E)), and seven non-structural proteins(NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).5, 6 The West Nile viral NS3 protease is absolutelyessential (along with viral-encoded cofactor NS2B) for post-translational processing of a viralpolypeptide precursor in infected host cells. This polypeptide provides the structural and functional viral proteins, and inhibition of its processing could represent a potential treatment for viral infections. Thus, this protease has become one of the key targets for developing anti-WNV drugs.1,7
The 440 West Nile Virus Protease Assay Kit provides a convenient, homogeneous assay for high throughput screening of West Nile Virus protease NS3 inhibitors. It allows for continuous quantification of protease activity using a fluorogenic peptide described in the literature.1 This peptide, Pyr-RTKR-AMC generates the AMC (7-amino-4-methylcoumarin) fluorophore upon NS3 protease cleavage. AMC has bright blue fluorescence that can be detected with excitation at 354nm and emission at 442nm.
Kit Components:Component A Microtiter Plate 1x500 wells Pyr-RTKR-AMC, WNV protease substrate, Ex/Em=354 nm/442nm 5mM DMSO solution, 1x250ulComponent B AMC, fluorescence reference standard, Ex/Em=354nm/442nm5 mM DMSO solution, 1x10ulComponent C Assay Buffer 2 x 50mlComponent D WNV Protease Inhibitor undeca-D-Arg-NH2 1 mM DMSO solution, 1x10ul
Materials Required (but not provided):
WNV protease: WNV protease can be produced in an E. coli expression system.1, 2 (active recombinant WNV protease available)
96-Well microplate: Black microplates can provide better signal to noise ratio.
Fluorescence microplate reader: Capable of detecting emission at 442nm with excitation at 354nm.
Storage and Handling:
Store all kit components at -20°C.
Protect Components A and B from light and from moisture.
Component C can be stored at room temperature for convenience.
