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| Product Name | Trypsin Activity Colorimetric Assay Kit |
| Catalog No. | ETR-HMM-0065 |
| Description | A colorimetric enzymatic assay for measuring trypsin and trypsin-like serine protease activity using the synthetic chromogenic substrate Nα-benzoyl-L-arginine p-nitroanilide (L-BAPNA). Trypsin cleaves the amide bond at the p-nitroanilide leaving group of the substrate, releasing yellow p-nitroaniline measured at 405 nm. |
| Intended Use | Quantifying trypsin activity in pancreatic homogenates, cell culture dissociation reagents, bioreactor media, and processed food samples; measuring serine protease activity in digestive disease research and enzyme quality control applications. |
| Principle / Technology | Trypsin (EC 3.4.21.4) cleaves amide and ester bonds on the C-terminal side of arginine and lysine residues; the synthetic substrate L-BAPNA mimics the recognition sequence and is hydrolyzed at the amide bond to release p-nitroaniline (molar absorptivity ~9,500 M-1cm-1 at 405 nm) and benzoyl-L-arginine; the rate of p-nitroaniline release is directly proportional to trypsin activity. |
| Detection Method | Colorimetric kinetic absorbance at 405 nm using a microplate reader or standard spectrophotometer |
| Sample Type | Pancreatic tissue homogenates, trypsin solution preparations, cell culture supernatants, digestive fluid samples, pancreatic juice, small intestine tissue extracts |
| Performance Range / Specifications | Trypsin activity range: 0.001–2 U/mL L-BAPNA units; one unit defined as amount hydrolysizing 1 μmol L-BAPNA per minute at 25°C pH 8.2; linear rate over 5–10 minutes |
| Sensitivity / LOD | Lower detection limit: approximately 0.1 μU/mL in buffered aqueous sample with 200 μL sample volume |
| Specificity | L-BAPNA has stringent specificity for trypsin and trypsin-like serine proteases (including thrombin, plasmin, and Factor Xa) through recognition of the arginine-based recognition sequence; subtilisin, papain, and other non-serine proteases show minimal activity; chymotrypsin activity can be distinguished using the alternative substrate Suc-AAPF-pNA |
| Reaction Conditions / Protocol | Prepare standard dilutions of trypsin (0.001–2 U/mL) in Tris buffer pH 8.2 with calcium chloride; add 10 μL sample or standard to microplate; add 200 μL pre-warmed L-BAPNA Substrate Solution (1 mM final in Tris-HCl pH 8.2 with CaCl2) to initiate reaction at 37°C; measure absorbance at 405 nm kinetically every minute for 10 minutes; calculate initial linear rate (ΔA405/min); convert to activity using ε = 9,500 M-1cm-1 |
| Components / Formulation | L-BAPNA Substrate (lyophilized, 50 μmol vials), Tris-HCl Assay Buffer pH 8.2, CaCl2 Solution (10 mM cofactor), Trypsin Standard (TPCK-treated bovine pancreatic trypsin, lyophilized), Trypsin Inhibitor (soybean trypsin inhibitor solution, 10 mg/mL, for specificity control) |
| Storage Conditions | Substrate at -20°C desiccated for 12 months; Trypsin Standard at -20°C single-use aliquots; Assay Buffer at 2–8°C; CaCl2 at room temperature |
| Shelf Life | 12 months from date of manufacture |
| Package Specifications | 200 assays, 500 assays (96-well format) |
| Product Form | Lyophilized substrate and enzyme standard with liquid assay buffers |
| Quality Control | Each lot tested with TPCK-trypsin reference standard; specific activity within ±15% of nominal value; inhibition by 0.1 mg/mL SBTI >98% confirming trypsin-specific reaction; substrate blank absorbance rate <0.001 ΔA/min |
| Key Features | L-BAPNA is the internationally standardized trypsin substrate enabling cross-study comparison; SBTI inhibitor control validates assay specificity; includes TPCK-treated reference trypsin for activity normalization; simple protocol with standard 405 nm detection wavelength |
For research use only, not for clinical use.
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