Recombinant Human CD39L4/ENTPD5 Protein, CF

• Assay Buffer: 25 mM Tris, 5 mM CaCl₂, pH 7.5
• Recombinant Human CD39L4/ENTPD5 (rhCD39L4) (Catalog # 5297-EN)
• Substrate: GDP (Sigma, Catalog # G-7127), 100 mM stock in deionized water
• 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 rhCD39L4 to 0.04 µg/mL in Assay Buffer.
2. Prepare a standard curve from 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock (this is the first dilution to use as a standard).
3. Perform six additional one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
4. Load 25 µL of 0.04 μg/mL rhCD39L4 and the standard curve into a plate. Include a Substrate Blank containing Assay Buffer.
5. Dilute the Substrate to 100 µM in Assay Buffer.
6. Add 25 µL of the 100 µM Substrate to all wells and mix well.
7. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
8. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
9. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
10. Read plate at 620 nm (absorbance) in endpoint mode.
11. Calculate specific activity:

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

• rhCD39L4: 0.001 µg
• Substrate: 35.7 µM