Tos-Gly-Pro-Arg-AMC . HCl

This protocol is only for experimental guidance. The actual application must be modified according to your actual needs.
Trypsin-like activity test in colon supernatant^[1][2].
1. Solution preparation
Substrate solution: Dissolve Tos-Gly-Pro-Arg-AMC . HCl in 50mM Tris-HCl (pH 8.0) buffer to prepare a 10mM stock solution. Dilute to a working concentration of 100µM before use and preheat to room temperature.
2. Sample preparation
A: Take 50mg of distal colon sample, rinse with Krebs solution, freeze quickly and store at -80°C.
B: Use liquid nitrogen to pulverize the sample to maintain enzyme activity, add lysis buffer to dissolve for 15min, centrifuge and collect the supernatant for use.
3. Fluorescence detection
A: Place the colon supernatant in a cold microtiter plate, ensuring that the sample remains cold to avoid loss of enzyme activity.
B: Add substrate solution preheated to room temperature and incubate for 20min.
C: Use an ELISA reader to dynamically detect the fluorescence intensity changes of AMC under the conditions of excitation wavelength 380nm and emission wavelength 460nm, and calculate the enzyme activity.
Notes:
1) For your safety and health, please wear lab coats and protective gloves when operating.
2) Enzyme activity experiments are affected by factors such as ion concentration, reaction temperature and pH. It is recommended to explore the optimal concentration when using for the first time.

References:
[1]. Ceuleers H, Hanning N, Heirbaut J, et al. Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post?inflammatory rat model for irritable bowel syndrome[J]. British Journal of Pharmacology, 2018, 175(17): 3516-3533.
[2]. Corvo I, Cancela M, Cappetta M, et al. The major cathepsin L secreted by the invasive juvenile Fasciola hepatica prefers proline in the S2 subsite and can cleave collagen[J]. Molecular and biochemical parasitology, 2009, 167(1): 41-47.