TACE (tumor necrosis factor-alpha converting enzyme), α-secretase, or ADAM17 is a member of the ADAM family of proteases that contain both disintegrin and metalloprotease (catalytic) domains. This enzyme is involved in sperm-egg binding and fusion, muscle cell fusion, neurogenesis, modulation of Notch receptor and ligand processing, and processing of the pro-inflammatory cytokine, TNFα. TACE is currently being explored as a target for anti-inflammatory drugs. SensoLyte® 520 TACE Activity Assay Kit uses a 5-FAM (fluorophore) and QXL™ 520 (quencher) labeled FRET peptide substrate for continuous measurement of enzyme activity. In the intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL™ 520. Upon cleavage of the FRET peptide by the active enzyme, 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, 5-FAM/QXL™ 520 based FRET peptide has less interference from the autofluorescence of test compounds and cellular components and provides better assay sensitivity. The kit can be used to detect the activity of TACE or screen for TACE inhibitors. The assays are performed in a convenient 96-well format.
Component A: QXL™ 520/5-FAM, TACE substrate, Ex/Em=490 nm/520 nm: 1 mM, 50 µL
Component B: 5-FAM, fluorescence reference standard, Ex/Em=490 nm/520 nm: 1 mM, 10 µL
Component C: Assay Buffer: 20 mL
Component D: Inhibitor of TACE: 1 mM, 10 µL
Component E: Stop Solution: 10 mL
Storage & stability
Store all kit components at -20°C. Protect Components A and B from light and from moisture. Components C and E can be stored at room temperature for convenience.
A role for cGMP in inducible nitric-oxide synthase (iNOS)-induced tumor necrosis factor (TNF) α-converting enzyme (TACE/ADAM17) activation, translocation, and TNF receptor 1 (TNFR1) shedding in hepatocytes