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 B is a member of the cathepsin family consisting of 12 cysteine proteases with broad exo- and endopeptidase activity. Elevated levels of cathepsin B were detected in metastases and neurological disorders including Alzheimer's disease (AD). The SensoLyte® 520 Cathepsin B Activity Assay Kit provides a QXL™ 520/HiLyte™ Fluor 488 labeled FRET peptide substrate for 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 nm/520 nm. With superior fluorescence quantum yield and longer emission wavelength, the QXL™ 520/HiLyte™ Fluor 488 based FRET peptide has less interference from the autofluorescence of test compounds and cellular components and provides better assay sensitivity.
Component A: QXL™520/HiLyte™ Fluor 488 Cathepsin B substrate, Ex/Em=490 nm/520 nm upon cleavage: 2 mM, 50 µL
Component B: HiLyte™ Fluor 488, fluorescence reference standard, Ex/Em=490 nm/520 nm: 1 mM, 10 µL
Component C: Cathepsin B enzyme, human liver: 5 µL
Component D: Assay Buffer: 20 mL
Component E: Cathepsin B inhibitor Ac-LVK-CHO: 100 µM, 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.