Recombinant Mouse Neutrophil Elastase/ELA2 Protein, CF

• Activation Buffer: 50 mM MES, 50 mM NaCl, pH 5.5
• Assay Buffer: 50 mM Tris, 1 M NaCl, 0.05% (w/v) Brij-35, pH 7.5
• Recombinant Mouse Neutrophil Elastase/ELA2 (rmELA2) (Catalog # 4517-SE)
• Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
• Substrate: MeOSuc-Ala-Ala-Pro-Val-AMC (Bachem, Catalog # I-1270), 10 mM in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmELA2 to 50 µg/mL in Activation Buffer containing 50 µg/mL rmCathepsin C.
2. Incubate for 2 hours at 37 °C to activate rmELA2.
3. Dilute active rmELA2 to 1 ng/µL in Assay Buffer.
4. Dilute Substrate to 200 µM in Assay Buffer.
5. Load into a plate 50 µL of 1 ng/µL rmELA2, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 200 µM Substrate.
6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

• rmELA2: 0.05 µg
• Substrate: 100 µM