Recombinant Human ADP-sugar Pyrophosphatase/NUDT5, CF

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

• Assay Buffer: 25 mM Tris, 5 mM MgCl₂, pH 7.5
• Recombinant Human ADP‑Sugar Pyrophosphatase/NUDT5 (rhNUDT5) (Catalog # 6414-NH)
• Coupling Enzyme: 5'-Nucleotidase/CD73 (rhCD73) (Catalog # 5795-EN)
• Substrate: Adenosine 5’-diphosphate ribose (ADPR) (Sigma, Catalog # A0752), 20 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 Substrate to 2.5 mM in Assay Buffer.
2. Dilute rhCD73 to 3.33 µg/mL in Assay Buffer.
3. Dilute rhNUDT5 to 0.667 µg/mL in Assay Buffer.
4. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water 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.
5. Prepare standard curve by performing seven 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.
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 15 µL of 0.667 µg/mL rhNUDT5 into the plate. Include a Control containing 15 µL of Assay Buffer.
8. Add 15 µL of 3.33 µg/mL rhCD73 to the wells, excluding the standard curve and the curve blank.
9. Start the reaction by adding 20 µL of 2.5 mM Substrate to the wells, exluding the standard curve and the curve blank.
10. Cover the plate with parafilm or a plate sealer and incubate at room temperature for 30 minutes.
11. Add 30 µL of the Malachite Green Reagent A to all wells.
12. Add 100 µL of deionized water to all wells.
13. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
14. Read plate at 620 nm (absorbance) in endpoint mode.
15. Calculate specific activity:

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

• rhNUDT5: 0.01 µg
• rhCD73: 0.05 µg
• Substrate: 0.238 mM