Recombinant Human QPCT Protein, CF

• Assay Buffer: 25 mM HEPES, pH 7.0
• rhPGPEP-1 Buffer: 0.1 M Tris, 5 mM DTT, pH 9.0
• Recombinant Human Glutaminyl‑peptide Cyclotransferase/QPCT (rhQPCT) (Catalog # 6368-ZN)
• Substrate: Q-AMC (Bachem, Catalog # I-1175)
• Recombinant Human PGPEP-1 (rhPGPEP-1) (Catalog # 6278-CY)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhQPCT to 0.2 μg/mL in Assay Buffer.
2. Dilute Substrate to 200 μM in Assay Buffer.
3. Combine 150 μL of 0.2 ug/mL rhQPCT and 150 μL of 200 μM Substrate. Include a Substrate Blank containing 150 μL Assay Buffer and 150 μL of 200 μM Substrate.
4. Incubate reaction at room temperature for 20 minutes.
5. Stop reaction by boiling at 100 °C for 5 minutes and cool on ice for 3 minutes.
6. Dilute rhPGPEP-1 to 1 μg/mL in rhPGPEP-1 Buffer.
7. Add 300 μL of 1 μg/mL rhPGPEP-1 to each vial.
8. Incubate reaction at room temperature for 10 minutes.
9. Load 100 μL in quadruplicate from each vial into a 96 well plate.
10. Read plate at excitation and emission wavelengths of 380 nm and 460 nm, respectively, in endpoint mode.
11. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

• rhQPCT: 0.005 μg
• Substrate: 50 μM