Recombinant Human Methionine Aminopeptidase 1D Protein, CF

• Activation Buffer: 50 mM HEPES, 0.1 mM CoCl₂, 100 mM NaCl, pH 7.5
• Assay Buffer: 25 mM Tris, pH 8.0
• Recombinant Human Methionine Aminopeptidase 1D/MAP1D (rhMAP1D) (Catalog # 4714-ZN)
• Recombinant Human DPPIV/CD26 (rhDPPIV/CD26) (Catalog # 9168-SE)
• Substrate: H-Met-Gly-Pro-AMC (Catalog # ES017)
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhMAP1D to 40 µg/mL in Activation Buffer.
2. Dilute Substrate to 500 µM with Activation Buffer.
3. Combine equal volumes of 40 µg/mL rhMAP1D with 500 µM Substrate. Include a Substrate Blank containing Activation Buffer and 500 µM Substrate.
4. Incubate at room temperature for 1 hour in the dark.
5. Stop the reaction by heating for 5 minutes at 95 °C. Put on ice for 3 minutes. Centrifuge briefly in a microcentrifuge.
6. Dilute rhDPPIV/CD26 to 2 ng/µL in Assay Buffer.
7. Load 50 µL of 2 ng/µL rhDPPIV/CD26 into a plate, and add 50 µL of the rhMAP1D/Substrate mixture (include Substrate Blank).
8. Incubate plate at room temperature for 15 minutes in the dark.
9. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
10. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

Per Well:

• rhMAP1D: 1 µg
• rhDPPIV/CD26: 0.1 µg
• Substrate: 125 µM