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| Product Name | Matrix Metalloproteinase (MMP) Activity Fluorescent Assay Kit (Broad-Spectrum) |
| Catalog No. | ETR-HMM-0069 |
| Description | A fluorometric assay for measuring the proteolytic activity of matrix metalloproteinases using a fluorescence-resonance energy transfer (FRET) substrate containing a quenched fluorophore (7-methoxycoumarin, Mca) coupled to a quencher (Dnp). MMP cleavage of the peptide substrate disrupts the FRET pair, generating a dose-dependent increase in fluorescence. |
| Intended Use | Broad-spectrum MMP activity profiling in conditioned cell culture media, tissue extracts, and plasma samples for cancer invasion research, extracellular matrix remodeling studies, arthritis and fibrosis research, and metalloprotease inhibitor screening. |
| Principle / Technology | The FRET substrate contains a peptide backbone with an MMP-cleavable sequence flanked by a fluorophore (Mca: 7-methoxycoumarin-4-acetic acid) and a quencher (Dnp: dinitrophenyl) in close proximity; in the intact substrate, the quencher suppresses Mca fluorescence through intramolecular energy transfer; upon peptide cleavage by active MMP at the Gly-Leu or Pro-Leu scissile bond, the fluorophore and quencher are separated, resulting in fluorescence dequenching proportional to proteolytic activity. |
| Detection Method | Fluorescence measurement (Ex/Em: 320–330/390–400 nm for Mca group) using a fluorescence microplate reader with UV capability |
| Sample Type | Conditioned culture medium from cancer cells, synoviocytes, or fibroblasts; tissue extracts from tumors, joints, lung, or skin; human serum and plasma after activation with APMA (4-aminophenylmercuric acetate); recombinant or purified MMP enzyme preparations |
| Performance Range / Specifications | Fluorescence detection range: 0.01–10 μM cleaved Mca-fluorophore; MMP activity detectable in as little as 2.5 μg total protein from conditioned media; incubation 1–24 hours at 37°C depending on enzyme activity |
| Sensitivity / LOD | Detection of MMP activity in samples containing approximately 1–5 ng/mL active MMP enzyme (specific for each MMP isoform due to varying kcat/Km values for the FRET substrate) |
| Specificity | FRET substrates are preferentially cleaved by gelatinases (MMP-2, MMP-9), collagenases (MMP-1, MMP-8, MMP-13), and stromelysin (MMP-3) depending on the peptide cleavage sequence used; metalloprotease activity requires zinc coordination; inhibition by 1 mM EDTA or GM6001 (broad-spectrum MMP inhibitor) confirms metalloprotease specificity; serine and cysteine protease activities do not cleave the substrate under the assay conditions |
| Reaction Conditions / Protocol | Activate latent MMPs in samples with 1 mM APMA (37°C, 1 hour) if desired for total MMP potential measurement; mix 10 μL activated sample or recombinant MMP with 90 μL Substrate Working Solution (FRET substrate diluted in assay buffer, final 10–20 μM substrate); incubate at 37°C for 1–24 hours; measure fluorescence (Ex/Em 320/390 nm); include EDTA-inhibited blank control and Mca-free peptide fluorescence calibration |
| Components / Formulation | FRET Substrate Peptide (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 or similar broad-spectrum peptide, lyophilized, 5 μmol), MMP Assay Buffer (Tris-HCl, NaCl, CaCl2, ZnCl2, Brij-35 detergent, pH 7.5), APMA Activator for pro-MMP activation, EDTA Inhibitor (negative control), Mca fluorescence calibration standard, MMP-2 or MMP-9 positive control (recombinant, partially purified) |
| Storage Conditions | FRET substrate lyophilized: -20°C protected from light and moisture for 12 months; DMSO stock: -20°C desiccated; Assay Buffer at 2–8°C; APMA at -20°C |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 50 assays, 200 assays (96-well fluorometric format) |
| Product Form | Lyophilized FRET substrate with separate liquid assay buffer, activation, and control components |
| Quality Control | Each lot tested against recombinant active MMP-2 and MMP-9 reference standards; substrate cleavage rate within ±20% of reference; EDTA inhibition >95%; fluorescence dequenching ratio (cleaved/intact substrate) ≥20× for positive signal validation |
| Key Features | FRET substrate provides very low background fluorescence without enzyme; broad-spectrum MMP detection using a single substrate; APMA activation allows differentiation between active and latent total MMP activity; recombinant MMP controls enable activity unit calculation; compatible with inhibitor IC50 determinations |
For research use only, not for clinical use.
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