Recombinant Human Meprin alpha Subunit/MEP1A Protein, CF

• Activation Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, pH 7.5
• Recombinant Human Meprin  alpha  Subunit/MEP1A (rhMEP1A) (Catalog # 3220-ZN)
• Activator: Trypsin (Sigma, Catalog # T-1426)
• AEBSF (Tocris, Catalog # 5175), 100 mM stock in deionized water
• Fluorogenic Peptide Substrate: MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-OH (Catalog # ES007)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhMEP1A to 100 µg/mL in Activation Buffer with 0.1 µg/mL Trypsin.
2. Incubate 100 µg/mL rhMEP-1A at 37 °C for 3 hours.
3. Stop Trypsin activity by adding AEBSF to a final concentration of 1 mM.
4. Dilute activated rhMEP1A to 0.4 µg/mL in Assay Buffer.
5. Dilute Substrate to 40 µM in Assay Buffer.
6. Load into a black well plate 50 µL of 0.4 µg/mL rhMEP1A, and start the reaction by adding 50 µL of 40 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 40 µM Substrate.
7. Read excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
8. Calculate specific activity:

     *Adjusted for Substrate Blank.

     **Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).

Per Well:

• rhMEP1A: 0.02 µg
• Substrate: 20 µM