Recombinant Mouse Meprin beta Subunit/MEP1B Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Activation Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% (v/v) Brij-35, pH 7.5 (TCNB)
• Assay Buffer: 50 mM Tris, pH 8.0
• Recombinant Mouse Meprin  beta  Subunit/MEP1B (rmMEP-1B) (Catalog # 3300-ZN)
• Trypsin (Sigma, Catalog # T-1426)
• AEBSF (Catalog # EI001) (100 mM stock in water)
• Substrate MCA-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys(Dnp)-Arg-Arg-NH₂ (Catalog # ES004)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmMEP-1B to 200 µg/mL in Activation Buffer.
2. Dilute Trypsin to 20 µg/mL in Activation Buffer.
3. Mix equal volumes of rmMEP and Trypsin for final concentrations of 100 µg/mL and 10 µg/mL respectively.
   Incubate at 37 ºC for 2 hours.
4. Stop reaction by adding AEBSF to a final concentration of 1 mM.
5. Dilute rmMEP-1B to 0.025 ng/µL in Assay Buffer.
6. Dilute Substrate to 20 µM in Assay Buffer.
7. Load 50 µL of 0.025 ng/µL rmMEP-1B in the plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing Assay Buffer and Substrate.
8. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
9. Calculate specific activity:

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

• rmMEP1B: 0.00125 µg
• Substrate: 10 µM