MeOSuc-Ala-Ala-Pro-Val-AMC

MeOSuc-Ala-Ala-Pro-Val-AMC Fluorescence Method for Determining Elastase Activity^[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Dissolve MeOSuc-Ala-Ala-Pro-Val-AMC in dimethyl sulfoxide (DMSO) to prepare a stock solution, and store at -40°C. Before use, dilute the DMSO stock solution approximately 100-fold into the reaction buffer to achieve a final concentration of 400μM MeOSuc-Ala-Ala-Pro-Val-AMC, or adjust according to the requirements of kinetic experiments.

2. Preparation of test solution: Collect the supernatant after cell lysis, or prepare the working concentration of elastase (e.g., porcine pancreatic elastase; PPE; 50nM), and equilibrate at 37°C for 10 minutes before use for subsequent detection.

3. Activity Assay:Rapidly mix 50μL of 400μM MeOSuc-Ala-Ala-Pro-Val-AMC with 50μL of the test solution in a 96-well clear bottom plate. Immediately record the fluorescence change over time using a fluorescence microplate reader (excitation/emission wavelengths = 355-380/440-460nm).
Precautions:
(1) For your safety and health, please wear a lab coat and disposable gloves during operation.

References:
[1] Ivanov D, Emonet C, Foata F, et al. A serpin from the gut bacterium Bifidobacterium longum inhibits eukaryotic elastase-like serine proteases. J Biol Chem. 2006 Jun 23;281(25):17246-17252.