Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated proteins capable of digesting extracellular matrix components. MMP-12 (macrophage elastase) is expressed in activated macrophages and may play a role in smoke-induced emphysema. SensoLyte® 520 MMP-12 Assay Kit uses a 5-FAM (fluorophore) and QXL520™ (quencher) labeled FRET peptide substrates for continuous measurement of the enzyme activities. This substrate showed excellent specificity to MMP-12 with minimal cross-reaction with MMP-1, 2, 7, 8, 9, 9, 13 and 14. In an intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL520™. Upon the cleavage of the FRET peptide by MMP-12, the fluorescence of 5-FAM is recovered, and can be continuously monitored at excitation/emission = 490 nm/520 nm. With superior fluorescence quantum yield and longer emission wavelength, 5-FAM/QXL520™ based FRET peptide is less interfered by the autofluorescence of test compounds and cellular components and provides better assay sensitivity.
Component A: MMP-12 substrate Ex/Em=490 nm/520 nm upon cleavage: 60 µL
Component B: 5-FAM-Pro-Leu-OH, Fluorescence reference standard Ex/Em=490 nm/520 nm: 1 mM, 10 µL
Component C: APMA, 4-aminophenylmercuric acetate (Caution: Organic mercury. Handle with care! Do not dispose into sink!): 1 M, 20 µL
Component D: Assay buffer: 20 mL
Component E: Stop solution: 10 mL
Storage & stability
Store all components at -20°C. Protect components A and B from light. Components D and E can be stored at 4 °C for convenience.