Cathepsins are a class of globular lysosomal proteases playing a vital role in mammalian cellular turnover. They degrade polypeptides and are distinguished by their substrate specificities. Cathepsin G is the serine protease released by neutrophils upon their activation. Cathepsin G is currently being explored as a target for anti-inflammatory and anti-infective drugs. The SensoLyte® 520 Cathepsin G Activity Assay Kit uses a HiLyte™ Fluor 488/QXL™ 520 FRET peptide substrate for the measurement of enzyme activity. In the intact FRET peptide, the fluorescence of HiLyte™ Fluor 488 is quenched by QXL™ 520. Upon cleavage of the FRET peptide by the active enzyme, the increase of fluorescence can be continuously monitored at excitation/emission = 490/520 nm. With superior fluorescence quantum yield and longer emission wavelength, the HiLyte™ Fluor 488/QXL™ 520 based FRET peptide has less interference from the autofluorescence of test compounds and cellular components and provides better assay sensitivity. The fluorescent signal from HiLyte Fluor™ 488 is stable at low pH, which is the optimal pH for cathepsin activity. The kit can be used to detect the activity of Cathepsin G enzyme in biological samples and purified enzyme preparations.
Component A: QXL™ 520/HiLyte™ Fluor 488, Cathepsin G substrate, Ex/Em=490 nm/520 nm upon cleavage: 55 µL
Component B: HiLyte™ Fluor 488, fluorescence reference standard, Ex/Em=490 nm/520 nm: 200 µM, 10 µL
Component C: Cathepsin G, Human Neutrophil: 0.1 mg/mL, 15 µL
Component D: Assay Buffer: 25 mL
Component E: Cathepsin G Inhibitor: 1 mM, 20 µL
Storage & stability
Store component C at -80°C. Store all other components at -20°C. Component D can be stored at room temperature for convenience. Protect components A and B from light and moisture.