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| Product Name | Trypsin Inhibitor Screening Kit (Fluorometric) |
| Catalog No. | ETR-HMM-0085 |
| Description | Fluorometric assay for screening trypsin inhibitors using a fluorogenic peptide substrate (Boc-Gln-Ala-Arg-AMC). Active trypsin cleaves the substrate releasing fluorescent 7-amino-4-methylcoumarin (AMC) detected at Ex/Em 380/460 nm. Inhibition of trypsin activity by test compounds reduces the fluorescence signal, enabling high-throughput screening of natural product extracts, synthetic compound libraries, and purified trypsin inhibitors like soybean trypsin inhibitor (SBTI) and aprotinin. |
| Intended Use | High-throughput screening of trypsin inhibitors; IC50 determination for inhibitor candidates; natural product extract screening for protease inhibitors; quality control of recombinant trypsin preparations; quantification of trypsin inhibitor content in plant and food samples. |
| Principle / Technology | Trypsin cleaves Boc-Gln-Ala-Arg-AMC → Boc-Gln-Ala-Arg + AMC (fluorescent); inhibitors reduce AMC release proportionally; IC50 calculated from dose-response curve of inhibitor concentration vs. residual activity |
| Detection Method | Fluorometric; Ex 355-380 nm, Em 440-460 nm; kinetic or endpoint mode; 96-well or 384-well plate format |
| Sample Type | Test compounds (in DMSO or aqueous buffer, final DMSO <2%); purified inhibitors; plant/seed extracts; trypsin inhibitor products; 1-10 µL sample per 100 µL assay |
| Sensitivity / LOD | Z' factor >0.7 for HTS (384-well); detects trypsin activity from 0.1 ng/mL; inhibitor IC50 reproducible within ±10% |
| Specificity | Trypsin-specific tripeptide substrate (Boc-Gln-Ala-Arg-AMC); minimal cross-reactivity with chymotrypsin, thrombin, or other serine proteases |
| Reaction Conditions / Protocol | Pre-incubate trypsin with test inhibitor (10-15 min at 25 °C); add fluorogenic substrate (final 50 µM); read fluorescence kinetically (Ex/Em 380/460 nm) for 10-30 min; calculate residual activity relative to uninhibited control |
| Components / Formulation | Trypsin (bovine pancreas, TPCK-treated, lyophilized, 1 mg), fluorogenic substrate (Boc-Gln-Ala-Arg-AMC, 10 mM in DMSO), trypsin assay buffer (Tris-HCl 50 mM, CaCl₂ 20 mM, pH 8.0), soybean trypsin inhibitor (SBTI, positive control inhibitor, 1 mg), AMC calibration standard (1 mM), DMSO for compound dissolution, black 96-well plate (optional) |
| Storage Conditions | -20 °C (trypsin, SBTI, substrate, AMC standard); assay buffer at 4 °C; protect fluorogenic materials from light |
| Shelf Life | 12 months at -20 °C; substrate DMSO stock 6 months at -20 °C protected from light |
| Package Specifications | 100 assays, 500 assays (96-well); 2,000 assays (384-well HTS) |
| Product Form | Lyophilized enzyme and inhibitor; liquid fluorogenic substrate in DMSO |
| Key Features | Fluorometric readout for HTS; Z' >0.7; includes SBTI positive control inhibitor; AMC standard for absolute quantification; trypsin and substrate concentrations optimized for inhibitor screening; compatible with plant extracts and compound libraries; DMSO-tolerant (up to 2% final) |
| Purity | Trypsin TPCK-treated, specific activity >10,000 U/mg; substrate >98% HPLC; AMC standard >99%; SBTI >95% SDS-PAGE |
| Concentration | As specified per kit; calibrator and substrate concentrations traceable to reference |
| Activity / Unit Definition | One unit defined as the amount of enzyme that catalyzes conversion of 1 µmol substrate per minute under specified conditions |
| Molecular Weight | Varies by target enzyme as documented |
| Source / Origin | Recombinant or purified from natural sources; synthetic chromogenic/fluorogenic substrates |
| pH Range / Optimal pH | pH 6.0–9.0 depending on specific enzyme assay |
| Shipping Conditions | Cold pack -20 °C; protect AMC reagents from light |
| Expiration Date / Stability | 12 months at -20 °C; reconstituted trypsin 1 week at 4 °C; substrate DMSO stock 6 months at -20 °C; discard substrate if AMC peak increases at Ex/Em 380/460 nm (auto-hydrolysis) |
| Regulatory / Compliance | For laboratory and research use only; RUO; DMSO flammable; ISO 9001 |
| Compatibility | Compatible with standard microplate readers (absorbance, fluorescence, luminescence modes) |
| Recommended Buffer System | Tris or phosphate buffer optimized per enzyme assay |
| Application Notes / Precautions | Include positive control and inhibitor control in each assay; read within linear range; protect light-sensitive substrates |
| Batch-to-Batch Consistency | Enzyme activity and standard curve linearity within ±15% of reference lot |
For research use only, not for clinical use.
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