This plan only provides a guide, please modify it to meet your specific needs.
1.Solution Preparation
(1) Prepare stock solution: Equilibrate the cryopreserved solid to room temperature, use DMSO to dissolve Boc-Gln-Ala-Arg-AMC. HCl, and prepare a 5mM storage solution.
Note: Unused stock solution should be aliquoted and stored in the dark at -20°C or -80°C to avoid repeated freeze-thaw cycles.
(2) Prepare working solution: Before the formal experiment, dilute the stock solution to the desired working concentration, such as 200μM, using an appropriate buffer.
Note:Optimal working concentrations should be adjusted based on actual conditions or determined by consulting literature and performing gradient concentration trials. Working solutions must be prepared fresh.
2.Steps to Measure Protease-Released AMC from Synthetic Substrate[1] (Reference only)
(1) Prepare samples according to experimental requirements.
(2) The experiment was conducted in a total volume of 200 μL, containing 5 μL of concentrated sample, 5 μL of substrate Boc-Gln-Ala-Arg-AMC. HCl stock solution (5 mM), and 190 μL of Tris HCl (100 mM, pH 8.5, containing 100 μg/mL bovine serum albumin).
(3) Fluorescence spectrophotometry is used to measure the fluorescence of AMC released by the hydrolysis of Boc-Gln-Ala-Arg-AMC·HCl, with an excitation wavelength of 380 nm and emission wavelength of 460 nm.
3.Steps to Measure Pancreatic Trypsin Activity [2] (Reference only)
(1) Pancreas Preparation: Approximately 40-50mg of pancreas tissue is homogenized in 1mL of MOPS homogenization buffer (250 mM sucrose, 5 mM MOPS (pH 6.5), 1 mM MgSO₄) using a motorized homogenizer.
(2) Centrifugation: The homogenate is briefly centrifuged (1000 x g, 1min) to remove larger particles.
(3) Prepare Reaction Mixture: 20μL of the cleared homogenate is mixed with 30μL of assay buffer (50 mM Tris-HCl (pH 8.1), 150 mM NaCl, 1 mM CaCl₂, 0.1 mg/mL bovine serum albumin).
(4) Add Fluorescent Substrate: 150μL of 200μM Boc-Gln-Ala-Arg-AMC·HCl fluorescent substrate (dissolved in assay buffer) is added to the mixture.
(5) Measure Fluorescence Increase: The increase in fluorescence is tracked continuously for 3 min in a fluorescent plate reader at an excitation wavelength of 380 nm and an emission wavelength of 460 nm.
(6) Data Processing: The rate of substrate cleavage is expressed as relative fluorescent units per second (RFU/s) and is normalized to the total protein in the assay mix (RFU/s/mg protein).
Precautions: Boc-Gln-Ala-Arg-AMC·HCl is a substrate for enzymes, and appropriate temperature and time conditions for enzymatic reactions should be determined based on the characteristics and activity of the enzyme being studied.
References:
[1] Fang, Ye, Shuang, et al. 3‐Cl‐AHPC inhibits pro‐HGF maturation by inducing matriptase/HAI‐1 complex formation[J] Journal of Cellular & Molecular Medicine, 2018 Oct.
[2] Andrea Geisz, Miklós Sahin-Tóth , et al. A preclinical model of chronic pancreatitis driven by trypsinogen autoactivation[J] nature communications, 28 November 2018.
