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| Product Name | FRET-Based Protease Activity Assay Kit (Caspase and Serine Protease Substrates) |
| Catalog No. | ETR-HMM-0070 |
| Description | A fluorescence-resonance energy transfer (FRET)-based assay kit for measuring the activity of diverse protease classes using a panel of quenched fluorogenic substrates. The kit includes substrates selective for cysteine proteases (caspases), serine proteases (trypsin, chymotrypsin, elastase), and aspartyl proteases, enabling broad protease activity profiling. |
| Intended Use | Comprehensive protease activity screening in cell lysates and biological fluids for drug target validation, protease inhibitor IC50 determination, cancer biomarker research, and investigation of protease cascades in inflammation and cell death pathways. |
| Principle / Technology | Each FRET substrate contains a peptide recognition sequence flanked by a fluorescent donor (such as AFC: 7-amino-4-trifluoromethylcoumarin or AMC: 7-amino-4-methylcoumarin) and a quencher (such as EDANS/DABCYL or Dnp); in intact substrates, the quencher suppresses donor fluorescence by resonance energy transfer; protease cleavage separates the fluorophore from the quencher, restoring fluorescence proportional to protease activity. |
| Detection Method | Fluorescence measurement with UV excitation (Ex 360–400 nm, Em 490–520 nm for AMC substrates; Ex 320 nm, Em 395 nm for AFC substrates) using a fluorescence plate reader |
| Sample Type | Purified recombinant proteases, cell lysates from apoptotic or protease-overexpressing cells, tissue homogenates (liver, lung), conditioned culture medium, plasma and serum (for kallikrein, thrombin, and elastase measurement) |
| Performance Range / Specifications | Fluorescence detection range: 0.001–100 μM released fluorophore; sensitivity depends on protease type and substrate Km; proteolytic rate measurable in samples containing ≥1 ng/mL active protease |
| Sensitivity / LOD | Detection of protease activity in samples containing approximately 0.1–10 nM active protease depending on kcat/Km and substrate concentration |
| Specificity | FRET substrate selectivity is determined by the peptide recognition sequence; representative substrates: Ac-DEVD-AFC (caspase-3), Ac-YVAD-AFC (caspase-1), Suc-LLVY-AMC (chymotrypsin), Ac-RVRR-AFC (furin), Mca-RPPGFSAFK(Dnp) (angiotensin converting enzyme); each substrate exhibits high specificity when used at below-Km concentrations |
| Reaction Conditions / Protocol | Prepare reaction in 96-well black-walled plate; add 80 μL Assay Buffer (50 mM HEPES pH 7.4, 0.1% CHAPS, 10 mM DTT for cysteine proteases; or pH 7.0 phosphate buffer for serine proteases); add 10 μL sample or purified enzyme standard; warm to 37°C; add 10 μL selected substrate (100 μM final); mix briefly; begin fluorescence reading immediately in kinetic mode at 37°C every 1–2 minutes for 30–60 minutes; plot fluorescence versus time and calculate initial linear rate; convert to nmol substrate cleaved/min using AMC or AFC calibration curves |
| Components / Formulation | Panel of FRET substrates (6–10 pre-selected quenched fluorogenic peptides for different protease classes, lyophilized individually), AMC Calibration Standard (lyophilized), AFC Calibration Standard, Cysteine Protease Assay Buffer (HEPES, CHAPS, DTT), Serine Protease Assay Buffer (sodium phosphate, pH 7.0), Metalloprotease Buffer (with CaCl2, ZnCl2), Protease Inhibitor Set (E-64 for cysteine, PMSF for serine, EDTA for metalloproteases, pepstatin for aspartyl, positive control protease) |
| Storage Conditions | All substrates at -20°C desiccated protected from light for 12 months; calibration standards at -20°C; assay buffers at -20°C (with DTT aliquots) or 2–8°C (without DTT) |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 100 reactions per substrate type (typically 5–10 substrate panel), 384-well format available |
| Product Form | Lyophilized substrate panel with calibration standards and buffer systems |
| Quality Control | Each lot tested with reference proteases for each substrate: e.g., DEVD-AFC with recombinant caspase-3, LLVY-AMC with chymotrypsin; fluorescence response and Km/Vmax values confirmed within ±25% of reference values; quenching efficiency of intact substrate >90% |
| Key Features | Multi-substrate panel enables broad protease class profiling from a single kit; FRET design provides self-referencing signal with very low background; compatible with diverse sample types from biological fluids to purified enzymes; calibration standards enable fluorescence unit to molarity conversion; inhibitor set allows protease class assignment |
For research use only, not for clinical use.
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