Recombinant Human Methionine Aminopeptidase 1 Protein, CF

• Activation Buffer: 50 mM HEPES, 0.1 mM CoCl₂, 0.1 M NaCl, pH 7.5
• Assay Buffer: 25 mM Tris, pH 8.0
• Recombinant Human Methionine Aminopeptidase 1/METAP1 (rhMETAP1) (Catalog # 3537-ZN)
• Recombinant Human DPPIV/CD26 (rhCD26) (Catalog #  9168-SE)
• Substrate 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 rhMETAP1 to 4 µg/mL in Activation Buffer.
2. Dilute Substrate to 200 µM in Activation Buffer.
3. Combine equal volumes of 4 µg/mL rhMETAP1 and 200 µM Substrate. Include a Substrate Blank containing Activation Buffer in place of rhMETAP1.
4. Incubate reactions for 5 minutes at room temperature.
5. Stop reactions by heating at 95-100 °C for 5 minutes.
6. Cool reactions on ice for 3 minutes and centrifuge briefly.
7. Dilute rhCD26 to 2 ng/µL in Assay Buffer.
8. Load into a black well plate 50 µL of each incubated mixture and add 50 µL 2 ng/µL rhCD26 to each.
9. Incubate at room temperature for 10 minutes.
10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
11. Calculate specific activity using the following formula:

     *Adjusted for Substrate Blank

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

Per Well:

• rhMETAP1: 0.10 µg
• rhCD26: 0.10 µg
• Substrate: 50 µM