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 S is a cysteine proteinase involved in the pathogenesis of autoimmune diseases, atherosclerosis, cancer, obesity and related diseases. The SensoLyte® 520 Cathepsin S Activity Assay Kit uses a 5-FAM/QXL™ 520 labeled FRET peptide substrate for measurement of enzyme activity. In the intact FRET peptide, the fluorescence of 5-FAM 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 nm/520 nm. With superior fluorescence quantum yield and longer emission wavelength, the 5-FAM/QXL™ 520 based FRET peptide shows less interference from the autofluorescence of test compounds and cellular components and provides better assay sensitivity.
Component A: 5-FAM/QXL™ 520, Cathepsin S substrate, Ex/Em=490 nm/520 nm upon cleavage: 1.6 mM, 50 µL
Component B: 5-FAM, fluorescence reference standard, Ex/Em=490 nm/520 nm: 1 mM, 10 µL
Component C: Cathepsin S, human spleen: 0.1 mg/mL, 10 µL
Component D: Assay Buffer: 20 mL
Component E: Cathepsin S Inhibitor: 1 mM, 10 µL
Component F: DTT: 1 M, 100 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.