Suc-Ala-Ala-Pro-Ile-pNA

This protocol only provides a guideline, and should be modified according to your specific needs.
1. Solution preparation
 
(1) Stock solution: Dissolve Suc-Ala-Ala-Pro-Ile-pNA in DMSO to a final concentration of 10 mM.
Note: After unused storage solution is aliquoted, store it at -20℃ in the dark to avoid repeated freezing and thawing.
 
(2) Working solution: Dilute the stock solution with experimental buffer to the required working concentration, for example: 0.2 mM.
Note: Please adjust the optimal working concentration according to the actual situation or refer to the literature to set the gradient concentration by yourself. The working solution must be prepared and used immediately.
 
2. Sensitivity Determination of Suc-Ala-Ala-Pro-Ile-pNA to Protease Cleavage^[1] (from the literature, for reference only)
(1) Materials Preparation: Substrate: Suc-Ala-Ala-Pro-Ile-pNA, Active enzyme, PBS buffer, 96-well microtiter plate, Microplate reader
(2) Reaction system: Add the chromogenic substrate Suc-Ala-Ala-Pro-Ile-pNA (5 μl, final concentration 0.2 mM) and active enzyme (a few microliters, the amount depends on the enzyme activity) to each well, and add PBS to make the final volume reach 200 μl.
(3) Reaction conditions: Perform the reaction at a specific temperature, such as 20°C.
(4) Measure the absorbance spectrophotometrically at 405 nm using a microplate reader. Measure at the following time points: 0, 20, 40, 60, 120, 180, 240, 300, and 360 minutes. Control: Set up a blank well for each substrate without adding any enzyme.
(5) Data Collection and Analysis: Conduct reactions in triplicate. Subtract the blank measurement at each time point. Use the mean absorbance of the three reactions for plotting and analysis.
 
Notes:
This protocol provides guidance for determining sensitivity to protease cleavage using Suc-Ala-Ala-Pro-Ile-pNA. It can be adapted based on other literature and specific experimental requirements.
 
References:
[1] Fu Z, Akula S, Qiao C, et al. Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity[J]. PLoS One, 2021, 16(5): e0252624.