Recombinant Human Meprin beta Subunit/MEP1B Protein, CF

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

• 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 8.0
• Recombinant Human Meprin  beta  Subunit/MEP1B (rhMEP1B) (Catalog # 2895-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 rhMEP1B to 100 µg/mL with 1 µg/mL Trypsin in Activation Buffer.
2. Incubate for 45 minutes at 37 °C. Stop Trypsin activation by adding AEBSF at a final concentration of 1 mM in Activation Buffer and mixing well.
3. Dilute activated rhMEP1B to 0.4 µg/mL in Assay Buffer.
4. Dilute Substrate to 20 µM in Assay Buffer.
5. Load 50 µL of 0.4 µg/mL rhMEP1B into plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of Substrate.
6. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
7. Calculate specific activity:

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

• rhMEP1B: 0.020 µg
• Substrate: 10 µM