Recombinant Human ENPP-7/Alk-SMase Protein, CF

• Assay Buffer: 50 mM Tris, 10 mM Sodium Taurocholate (Sigma, Catalog # T4009), 150 mM NaCl, pH 9.0
• rhALPL Buffer: 50 mM Tris, 2 mM MgCl₂, pH 9.0
• Recombinant Human ENPP‑7/Alk‑SMase (rhENPP-7) (Catalog # 4924-EN)
• Substrate: Sphingomyelin (egg, chicken) (SPM) (Avanti Polar Lipids, Inc., Catalog # 860061C)
• Phosphocholine Chloride (Sigma, Catalog # P0378), 100 mM stock in deionized water
• Recombinant Human Alkaline Phosphatase/ALPL (rhALPL) (Catalog # 2909-AP)
• Malachite Green Phosphate Detection Kit (Catalog # DY996)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute SPM to 0.4 mM in Assay Buffer. Note: Heat buffer to 100 °C and then add SPM for proper solubilization. Vortex well.
2. Dilute rhENPP-7 to 0.2 µg/mL in Assay Buffer.
3. Mix 50 µL of SPM and 50 µL of rhENPP-7 to start the reaction. For blank, substitute SPM with Assay Buffer and keep on ice during incubation.
4. Incubate at 37 °C for 30 minutes.
5. Stop the reaction by heating all vials at 95‑100 °C for 6 minutes. Cool on ice for 1 minute followed by brief centrifugation.
6. Prepare a standard curve from the 100 mM Phosphocholine Chloride stock by adding 10 µL of the 100 mM Phosphocholine Chloride Standard to 990 µL of Assay Buffer for a 1 mM stock. Continue by adding 100 µL of the 1 mM Phosphocholine Chloride stock to 900 µL of Assay Buffer for a 100 µM stock (This is the first dilution to use as a standard). Complete the standard curve by preparing the following dilutions in Assay Buffer: 80, 60, 40, 20, 10, and 5 μM.
7. Dilute rhALPL to 4 µg/mL in rhALPL Buffer.
8. Combine 100 µL of 4 µg/mL rhALPL with each of the reaction mixture, Blank, and each dilution of the standard curve (100 μL). The total volume of all vials should be 200 µL.
9. Incubate at room temperature for 20 minutes.
10. Mix equal volumes of the Malachite Green Reagent A and B together for enough volume to add 80 µL of the Malachite green A and B mixture to all vials. Add the mixture to vials and vortex.
11. Incubate at room temperature for 10 minutes. Note: Do not let mixtures incubate longer that the recommended time, precipitation may occur.
12. Load 70 µL from each vial into a plate.
13. Read plate at 620 nm (absorbance) in endpoint mode.
14. Calculate specific activity:

     *Derived from the standard curve using linear or 4-parameter fitting and adjusted for Blank.

Per Well:

• rhENPP-7: 0.0025 µg
• Sphingomyelin: 0.071 mM
• Phosphocholine Chloride Curve: 0, 125, 250, 500, 1000, 1500, 2000, and 2500 pmol