Recombinant Human ENPP-2/Autotaxin Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 5 mM MgCl₂, 0.02% Brij-35 (w/v), pH 8.5
• Recombinant Human ENPP‑2/Autotaxin (rhENPP-2) (Catalog # 5255-EN)
• Substrate: Bis(p-Nitrophenyl) Phosphate Sodium Salt (BPNPP) (Sigma, Catalog # N3002), 40 mM stock in deionized water (Note: Heating may be necessary to solubilize Substrate.)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhENPP-2 to 0.1 ng/µL in Assay Buffer.
2. Dilute Substrate BPNPP to 2 mM in Assay Buffer.
3. Load into the wells of a microplate 50 µL of 0.1 ng/µL rhENPP-2 and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate.
4. Read at 400 nm (absorbance) in kinetic mode for 5 minutes.
5. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard p-Nitrophenol (Sigma-Aldrich, Catalog # 241326).

• rhENPP-2: 0.005 µg
• Substrate: 1 mM