Recombinant Cynomolgus Monkey CD39/ENTPD1 His 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: 25 mM Tris, 5 mM CaCl₂, pH 7.5
• Recombinant Cynomolgus Monkey CD39 (rcynoCD39) (Catalog # 11092-EN)
• Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
• Malachite Green Phosphate Detection Kit (Catalog # DY996)
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare a standard curve from the 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.
2. 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. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
4. Dilute rcynoCD39 to 0.04 µg/mL in Assay Buffer.
5. Dilute ATP to 400 µM in Assay Buffer.
6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
7. Load 25 µL of the 0.04 µg/mL rcynoCD39 into empty wells of the plate. Include a Control containing 25 µL of Assay Buffer.
8. Start the reactions by adding 25 µL of 400 µM ATP to all the wells, excluding the standard curve and curve blank.
9. Incubate sealed plate at 37 °C for 20 minutes.
10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
11. Add 100 µL of deionized water to all wells. Mix briefly.
12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
13. Read plate at 620 nm (absorbance) in endpoint mode.
14. Calculate specific activity:
    
    

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

• rcynoCD39: 0.001 µg
• ATP: 200 µM