β-Secretase (BACE1) is a membrane-anchored aspartic protease existing in acidic subcellular vesicles. It is a key player in producing β-amyloid peptides through proteolytic cleavage of the β-amyloid precursor protein (APP). Accumulation of neurotoxic β-amyloid peptide is seen in senile plaques in the brains of patients with Alzheimer’s disease (AD), an age-related cognitive disorder. Therefore, targeting β-secretase is considered a potential therapeutic strategy for the prevention and treatment of AD. The SensoLyte® 520 β-Secretase Assay Kit is a homogeneous assay that can be used to detect the activity of enzyme and for screening of secretase inhibitors. It contains a QXL® 520/ HiLyte™ Fluor 488 FRET substrate. The sequence of this FRET peptide is derived from the β-secretase cleavage site on the Swedish APP mutation. This mutation causes the enhancement of β-secretase to process APP, resulting in an early onset of AD. Active β-secretase cleaves FRET substrate into two separate fragments resulting in the release of HiLyte™ Fluor 488 fluorescence that can be monitored at excitation/emission= 490/520 nm. The long wavelength fluorescence of HiLyte™ Fluor 488 is less interfered by the autofluorescence of components in biological samples and test compounds.
Component A: HiLyte™ Fluor 488/QXL™-520 BACE1 substrate, Ex/Em=490/520 nm upon cleavage: 60µL
Component B: HiLyte™ Fluor 488, fluorescence reference standard 1 mM DMSO solution: 20 µL
Component C: BACE1 Inhibitor 250 µM: 10 µL
Component D: 2X Assay buffer: 20 mL
Component E: Stop Solution: 10 mL
Component F: Human BACE1 enzyme: 20 µL
Storage & stability
Store all kit components at -20°C, except for Component F. Store b-secretase (Component F) at –80°C. Protect Components A and B from light and from moisture. Components D and E can be stored at room temperature for convenience.