Matrix metalloproteinases (MMPs) belong to a family of secreted or membrane-associated proteins capable of digesting extracellular matrix components. The importance of MMPs in tumor development and invasion as well as other diseases is well known. The SensoLyte® 520 Generic MMP Assay Kit is optimized to detect the activity of a variety of MMPs, including MMP-1, 2, 3, 7, 8, 9, 12, 13, and 14. Compared to the SensoLyte® 520 kits designed for individual MMP, the Generic MMP Assay kit is 5 to 20 fold more sensitive, although less specific. This kit is ideal for detecting generic MMP activity in biological samples or for high throughput screening of MMPs’ inducers and inhibitors using purified MMPs. This kit uses a 5-FAM (fluorophore) and QXL520™ (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 SensoLyte®. Upon the cleavage of the FRET peptide by MMPs, 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. The assays are performed in a convenient 96-well or 384-well microplate format.
Component A: Generic MMP 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: 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.