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® 520 Thrombin Assay Kit is optimized for detecting thrombin activity. This kit contains a novel internally quenched 5-FAM/QXL™ 520 FRET substrate for thrombin. Thrombin cleaves the FRET substrate into two separate fragments resulting in the release of 5-FAM fluorescence which can be monitored at excitation/emission= 490 nm/520 nm. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples and test compounds. The assay can be performed in a convenient 96-well or 384-well microplate format for screening of enzyme inhibitors.
Component A: 5-FAM/QXL™520 Thrombin substrate, Ex/Em=490 nm/520 nm upon cleavage: 2 mM, 50 µL
Component B: 5-FAM, Fluorescence reference standard, Ex/Em=490 nm/520 nm: 2 mM, 10 µL
Component C: 2X Assay buffe:r 20 mL
Component D: Purified human thrombin enzyme: 0.1 mg/mL, 40 µL
Component E: Thrombin inhibitor: 60 µM, 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.