Recombinant Human Vanin-1/VNN1 Protein, CF

• Assay Buffer: 50 mM HEPES, 2 mM DTT, 1% Brij-35 (w/v), pH 7.0
• Recombinant Human Vanin-1/VNN1 (rhVNN1) (Catalog # 7999-AH)
• Substrate: Pantethine (Sigma, Catalog # P2125) 50 mM stock in deionized water
• o-pthalaldehyde (Sigma, Catalog # P0657) 50 mg/mL (373 mM) stock in DMSO
• 0.5 M Sodium Borate, pH 9.0
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent

1. Dilute rhVNN1 to 1 µg/mL in Assay Buffer.
2. Dilute Substrate to 500 µM in Assay Buffer.
3. Load 50 µL of dilute rhVNN1 to empty wells of a black well plate.
4. Start reaction by adding 50 µL of dilute substrate to wells containing enzyme. Create Enzyme Controls by not adding substrate to the wells.
5. Seal plate with a plate sealer and incubate at 37 °C for 30 minutes.
6. Prepare Detection mixture containing 15 mM oPA in 0.5 M sodium borate, pH 9.0.
7. Add 100 µL Detection mixture to all wells used. For Enzyme Controls, add dilute substrate after Detection Mixture is added.
8. Mix well and incubate sealed plate at room temperature for 5 minutes.
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 Enzyme Control
     **Derived using calibration standard Cysteamine Hydrochloride (Sigma, Catalog # M6500).

• rhVNN1: 0.050 μg
• Substrate: 125 µM