Recombinant Human Adenosine Kinase/ADK Protein, CF

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

• Assay Buffer: (10X) 250 mM HEPES, 1.5 M NaCl, 100 mM MgCl₂, 100 mM CaCl₂ pH 7.0 (supplied in kit)
• Recombinant Human Adenosine Kinase/ADK (Catalog # 8024-AK)
• Adenosine (Sigma, Catalog # A9251), 10 mM stock in deionized water
• Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water
• Universal Kinase Activity Kit (Catalog # EA004)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare 1X Assay Buffer by diluting 10X Assay Buffer in deionized water.
2. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X  Assay Buffer for a 100 µM stock.
3. Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
4. Load 50 µL of each dilution of the standard curve into a plate in triplicate. Include a curve blank containing 50 μL of 1X Assay Buffer.
5. Prepare Substrate Mixture composed of 0.364 mM ATP and 3.64 mM Adenosine in 1X Assay Buffer.
6. Dilute rhADK to 50 ng/µL in 1X Assay Buffer.
7. Load 20 µL of the 50 ng/µL rhADK into the plate in triplicate. Include a control containing 20 µL of 1X Assay Buffer.
8. Add 55 µL of Substrate Mixture to the wells, excluding the standard curve and curve blank.
9. Incubate sealed at 37 °C for 1 hour.
10. Dilute Coupling Phosphatase 4 (supplied in kit) to 2.67 µg/mL in 1X Assay Buffer.
11. Add 100 µL of 1X Assay Buffer to each well containing the standard curve and curve blank.
12. Add 75 µL of 2.67 µg/mL Coupling Phosphatase 4 to wells containing enzyme and blanks, excluding the standard curve and curve blank.
13. Incubate sealed plate at room temperature for 5 minutes.
14. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
15. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
16. Read plate at 620 nm (absorbance) in endpoint mode.
17. Calculate specific activity:

     *Derived from the phosphate standard curve using linear fitting and adjusted for Substrate Blank.
     **Use the sum of the incubation times prior to the addition of Malachite Green reagents due to the constant conditions throughout the assay.

Note:  No coupling rate is applicable for this assay.

• rhADK: 1.0 µg
• Coupling Phosphatase 4: 0.2 µg
• ATP: 20 nmol
• Adenosine: 200 nmol