Factor Xa (FXa) is a serine endopeptidase composed of two disulfide-linked subunits. FXa leads to blood clot formation by converting prothrombin to thrombin through the prothrombinase complex. FXa is generated from zymogen Factor X via the intrinsic and extrinsic pathways and is the rate-limiting step in the propagation of thrombin generation. In the presence of Ca2+ ions, FXa forms prothrombinase with factor Va on the phospholipid membrane of the activated platelets. FXa has emerged as an attractive target for drug discovery for thromboembolic diseases. The SensoLyte® Rh110 Factor Xa Assay Kit provides a convenient assay for screening of FXa inhibitors or continuous assay of enzyme activity using a fluorogenic substrate. Upon FXa protease cleavage, this substrate generates the Rh110 (rhodamine 110) fluorophore which has a bright green fluorescence that can be detected at excitation/emission=490 nm/520 nm. The longer-wavelength spectra and higher extinction coefficient of the Rh110 provide greater sensitivity and less interference from other reaction components.
Component A: Rh110 Factor Xa substrate, Ex/Em=490 nm/520 nm upon cleavage: 0.4 mM, 50 µL
Component B: Rh110, fluorescence reference standard, Ex/Em=490 nm/520 nm: 0.4 mM, 10 µL
Component C: Purified Bovine Factor Xa: 5 ng/µL, 20 µL
Component D: 2X Assay Buffer: 25 mL
Component E: Factor Xa Inhibitor: 1 mM, 20 µL
Storage & stability
Store all kit components at -20°C, except for Component C. Store Component C at -80°C. Protect Components A and B from light and moisture. Component D can be stored at room temperature for convenience.