Recombinant Mouse CD39/ENTPD1 Protein, CF

• Assay Buffer: 25 mM Tris, 5 mM CaCl₂, pH 7.5
• Recombinant Mouse CD39/ENTPD1 (rmCD39) (Catalog # 4398-EN)
• Substrate: Adenosine triphosphate (ATP) (Sigma, Catalog # A-7699), 10 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. Prepare a standard curve from the 1 M Phosphate Standard supplied in the malachite green phosphate detection kit. First, add 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock.
Then, add 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). Finally, perform six additional two-fold serial dilutions of the 100 µM phosphate stock. The standard curve has a range of 0.039 to 2.5 nmol per well.
2. Dilute rmCD39 to 0.02 µg/mL in Assay Buffer.
3. Transfer to a plate (in duplicate) 25 µL of standard curve, diluted rmCD39 at 0.02 µg/mL, and blanks (Assay Buffer).
4. Dilute Substrate to 100 µM in Assay Buffer.
5. Add 25 µL of the Substrate to all wells. Mix well.
6. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 30 minutes.
7. After incubation, add 10 µL of the Malachite Green Reagent A to each sample, standard, and blank. Mix and incubate for 10 minutes at room temperature.
8. Add 10 µL of the Malachite Green Reagent B to each sample, standard, and blank. Mix and incubate for 20 minutes at room temperature.
9. Read plate at 620 nm (absorbance) in endpoint mode.
10. Calculate specific activity:

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

Per Reaction:

• rmCD39: 0.0005 µg
• Substrate: 50 µM