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

• Assay Buffer: 50 mM Tris, 10 mM Sodium Taurocholate (Sigma, Catalog # T4009), 150 mM NaCl, pH 9.0
• rhTNAP Buffer: 50 mM Tris, 2 mM MgCl₂, pH 9.0
• Recombinant Mouse ENPP‑7/Alk‑SMase (rmENPP-7) (Catalog # 5160-EN)
• Substrate: Sphingomyelin (egg, chicken) (SPM) (Avanti Polar Lipids, Inc., Catalog # 860061C), 35 mM in chloroform
• Phosphocholine Chloride (Sigma, Catalog # P0378), 100 mM stock in deionized water
• Recombinant Human Alkaline Phosphatase/ALPL/TNAP (rhTNAP) (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 to help keep SPM solubilized. Vortex well.
2. Dilute rmENPP-7 to 0.1 µg/mL in Assay Buffer.
3. Mix equal volumes of diluted SPM and diluted rmENPP-7.
4. Incubate at 37 °C for 30 minutes. As a Substrate Control substitute SPM with Assay Buffer and keep on ice during incubation.
5. Stop the reaction by heating all vials at 95-100 °C for 6 min. Cool on ice for 1 min followed by brief centrifugation in microcentrifuge.
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).
7. Make the following serial dilutions from the 100 µM Phosphocholine Chloride stock in Assay Buffer to complete standard curve: 80, 60, 40, 20, 10 and 5 μM.
8. Dilute rhTNAP to 4 µg/mL in rhTNAP Buffer.
9. Combine 100 µL of the 4 µg/mL rhTNAP with 100 µL each of the diluted rmENPP-7/SPM mixture, the dilutions of the standard curve, and the Substrate Control.
10. Incubate at room temperature for 20 min.
11. Mix equal volumes of the Malachite Green Reagents A and B together for enough volume to add 80 µL of the mixture to all vials.
12. Add the Malachite Green Reagent A and B mixture to vials and vortex.
13. Incubate at room temperature for 10 min.  Note:  Do not let mixtures incubate longer, precipitation may occur.
14. Load 70 µL from each vial into a plate.
15. Read plate at 620 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

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

• rmENPP-7: 0.00125 µg
• Sphingomyelin: 0.071 mM
• Phosphocholine Chloride Curve: 0, 125, 250, 500, 1000, 1500, 2000, and 2500 pmol