Insulin degrading enzyme (IDE) is considered a physiological and pathological relevant enzyme. Growing evidences have indicated that IDE may be involved in the pathogenesis of Alzheimer’s disease and diabetes mellitus type 2. The SensoLyte® 520 IDE Activity Assay Kit can be used to detect enzyme activity in biological samples or in purified enzyme preparations. The unique FRET substrate was derived from an APP sequence designed to reduce cross reactivity with Neprilysin, ADAM10, TACE, BACE-1, and BACE-2. When active IDE cleaves the FRET substrate, it results in an increase of 5-FAM fluorescence, which is monitored at excitation/emission = 490 nm/520 nm. The long wavelength fluorescence of 5-FAM is also less interfered by the autofluorescence of components in biological samples and test compounds. This assay can detect as low as 0.8 ng/mL active IDE.
Component A: 5-FAM /QXL® 520 IDE substrate Ex/Em=490 nm/520 nm upon cleavage: 1 mM, 50 µL
Component B: 5-FAM fluorescence reference standard, Ex/Em=490 nm/520 nm: 1 mM, 12 µL
Component C: Human recombinant IDE: 0.1 mg/mL, 10 µL
Component D: Assay Buffer: 25 mL
Component E: Inhibitor: 1 M, 10 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.