Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated zinc endopeptidases capable of digesting extracellular matrix components. MMP-14 (MT1-MMP) is the membrane bound MMP. When expressed in cells, the catalytic and the hemopexin domains of MMP-14 become exposed on cell surfaces. This enzyme plays an important role in tumor invasion. SensoLyte® 520 MMP-14 Assay Kit uses a 5-FAM (fluorophore) and QXL™520 (quencher) labeled FRET peptide substrates for continuous measurement of the enzyme activities. In an intact FRET peptide, the fluorescence of 5-FAM is quenched by QXL™ 520. Upon the cleavage of the FRET peptide by MMP-14, 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/QXL™520 based FRET peptide is less interfered by the autofluorescence of test compounds and cellular components and provides better assay sensitivity.
Component A: MMP-14 substrate 5-FAM/QXL™520 FRET peptide Ex/Em=490nm/520nm 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.