Recombinant Mouse VAP-1 Protein, CF

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

• Assay Buffer: 50 mM HEPES, pH 7.5 
• Recombinant Mouse VAP‑1/AOC3 (rmVAP-1) (Catalog # 6107-AO)
• Coupling Agent: Horseradish Peroxidase (HRP) (250-330 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
• Substrate Component 1: Benzylamine (Sigma, Catalog # B5136), 100 mM stock in deionized water
• Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rmVAP-1 to 8 ng/µL in Assay Buffer.
2. Prepare the Substrate mixture 2 mM Benzylamine, 2 units/mL HRP and 100 µM AUR in Assay Buffer.
3. In a plate, load 50 µL of 8 ng/µL rmVAP-1 and start the reaction by adding 50 µL of the Substrate mixture (step 2). Include a Substrate Blank containing 50 µL of the Assay Buffer and 50 µL of the Substrate mixture.
4. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode for 5 minutes. Note: A manual cutoff must be set at a wavelength of 570 nm.
5. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 1 mM Benzylamine, and a curve of Hydrogen Peroxide (Sigma, Catalog # H1009) in Assay Buffer. Use this oxidized AUR curve to determine the conversion factor.

• rmVAP-1: 0.4 µg
• Benzylamine: 1 mM
• HRP: 1 unit/mL
• AUR: 50 µM