Thrombin, a serine protease, plays a central role in hemostasis by converting soluble plasma fibrinogen into an insoluble fibrin clot and by promoting platelet aggregation. Thrombin also influences a number of normal and pathological processes, including inflammation, tissue repair, embryogenesis, angiogenesis, and tumor invasion. The SensoLyte® AFC Thrombin Assay Kit is optimized for screening of enzyme inhibitors. This kit contains a fluorogenic substrate which shows high reactivity and low background. Thrombin cleaves the substrate resulting in the release of the AFC (7-amido-4-trifluoromethylcoumarin) fluorophore. Fluorescence can be monitored at excitation/emission= 380 nm/500 nm. The assays are performed in a convenient 96-well or 384-well microplate format.
Component A: AFC Thrombin substrate, Ex/Em=380 nm/500 nm: 5 mM, 50 µL
Component B: AFC, Fluorescence reference standard, Ex/Em=380 nm/500 nm: 5 mM, 10 µL
Component C: 2X Assay buffer: 20 mL
Component D: Purified human thrombin enzyme: 0.01 mg/mL, 40 µL
Component E: Thrombin inhibito:r 10 mM, 10 µL
Component F: Stop solution: 5 mL
Storage & stability
Store all kit components, except Component D, at -20°C. Store Component D at -80°C. Aliquot as needed to avoid multiple freeze-thaw cycles. Protect Components A and B from light and moisture. Components C and F can be stored at room temperature for convenience.