Recombinant Human Carboxypeptidase E/CPE 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.

• Assay Buffer: 50 mM NaOAc, 5 µM ZnCl₂, pH 5.5
• Recombinant Human Carboxypeptidase E/CPE (rhCPE) (Catalog # 3587-ZN)
• Substrate: Benzoyl-Ala-Arg-OH (Bachem, Catalog # G-4145), 50 mM stock in DMSO
• ortho-phthaldialdehyde (OPA) (134.13 g/mol) (Sigma, Catalog # P0657), 50 mg/mL stock in DMSO
• OPA Buffer: 0.2 M NaOH containing 0.1% mercaptoethanol (v/v)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCPE to 0.1 ng/µL in Assay Buffer.
2. Dilute Substrate to 1 mM in Assay Buffer.
3. Combine 75 µL of 0.1 ng/μL rhCPE and 75 µL of 1 mM Substrate. Include a Blank containing 75 µL of Substrate only.
4. Incubate for 10 minutes at room temperature.
5. Add 150 µL of 15 mM OPA in 0.2 M NaOH containing 0.1% mercaptoethanol (v/v) to reaction vials and the Blank (stops reaction).
6. After stopping the reaction, add 75 µL of 0.1 ng/µL rhCPE to the Blank.
7. Mix well and incubate at room temperature for 2-10 minutes.
8. Load 200 µL of sample(s) and blank into plate.
9. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively in endpoint mode.
10. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard L-Arginine (Sigma, Catalog # A-5006).

• rhCPE: 0.005 µg
• Substrate: 0.25 mM
• OPA: 7.5 mM