Recombinant Cynomolgus 5'-Nucleotidase/CD73 His Protein, CF

• Assay Buffer: 25 mM Tris, 5 mM MgCl₂, pH 7.5
• Recombinant Cynomolgus Monkey 5'‑Nucleotidase/CD73 (rcynoCD73) (Catalog # 10173-EN)
• Adenosine monophosphate (AMP) (Sigma, Catalog # A1752), 5 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. 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).
2. Perform six additional one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer.  The standard curve has a range of 0.078 to 5 nmoles per well.
3. Dilute Substrate to 100 µM in Assay Buffer.
4. Dilute rcynoCD73 to 0.08 µg/mL in Assay Buffer.
5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
6. Load 25 µL of 0.08 µg/mL rcynoCD73 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
7. Add 25 µL of diluted Substrate to all wells, excluding the standard curve. 
8. Seal plate and incubate at 37 °C for 20 minutes.
9. Add 30 µL of the Malachite Green Reagent A to all wells used, including standard curve.  Mix briefly.
10. Add 100 µL of deionized water to all wells used, including standard curve.  Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells used, including standard curve.  Mix briefly.
12. Seal plate and incubate at room temperature for 20 minutes.
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.

• rcynoCD73: 0.002 µg
• Substrate: 50 µM