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.1 Thrombin also influences a number of normal and pathological processes, including inflammation,2 tissue repair,3 embryogenesis,4 angiogenesis,5 and tumor invasion.6
The SensoLyte 520 Thrombin Activity Assay Kit kit employs a novel internally quenched 5-FAM/QXL 520 FRET substrate (Figure 1). Thrombin cleaves the FRET substrate into two separate fragments resulting in release of the fluorescence of 5-FAM, a green dye. Increase of fluorescence can be monitored at the commonly used excitation and emission wavelengths of 490 nm and 520 nm, respectively. The long wavelength fluorescence of 5-FAM is less interfered by the autofluorescence of components in biological samples and test compounds, resulting in a high signal to background ratio. Figure 2 shows the inhibition data.
AnaSpec also offers a shorter wavelength thrombin assay, which employs an AFC (7-amido-4-trifluoromethylcoumarin) labeled substrate, the SensoLyte AFC Thrombin Activity Assay Kit.
1. Crawley, JT. et al. J. Thromb. Hemost. 5, 95(2007).
2. Croce, K. et al. Curr. Opin. Hematol. 14, 55 (2007).
3. Artuc, M. et al. Exp. Dermatol. 15, 523 (2006).
4. Griffin, CT. et al. Science 293, 1666 (2001).
5. Maragoudakis, ME. et al. Biochem. Soc. Trans. 30, 173 (2002).
6. Snyder, KM. et al. Semin. Thromb. Hemost. 34, 734 (2008).
AnaSpec (anaspec.com) is a leading provider of integrated proteomics solutions to the world’s largest biotech, pharmaceutical, and academic research institutions. With a vision for innovation through synergy, AnaSpec focuses on three core technologies: peptides, detection reagents, and combinatorial chemistry.