Recombinant Human N-Acetylmannosamine Kinase/GNE 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 (10X): 250 mM HEPES, 1500 mM NaCl, 100 mM MgCl₂, 100 mM CaCl₂ pH 7.0 (supplied in kit)
• Recombinant Human N‑Acetylmannosamine Kinase/GNE (rhGNE) (Catalog # 8268-GK)
• N-Acetyl-D-mannosamine (Sigma, Catalog # A8176), 100 mM stock in deionized water
• Universal Kinase Activity Kit (Catalog # EA004)
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare 1X Assay Buffer by diluting 10X Assay Buffer using 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 0.4 mM ATP and 2 mM N-Acetyl-D-mannosamine in 1X Assay Buffer.
6. Dilute rhGNE to 66.67 μg/mL in 1X Assay Buffer.
7. Load 15 µL of the 66.67 μg/mL rhGNE into the plate in triplicate. Include a control containing 15 µL of 1X Assay Buffer.
8. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in 1X Assay Buffer.
9. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
10. Add 25 µL of Substrate Mixture to the wells, excluding the standard curve.
11. Incubate sealed plate at room temperature for 10 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
13. Add 100 µL of deionized water to all wells. Mix briefly.
14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
15. Read plate at 620 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

     *Derived from the phosphate standard curve using linear fitting and adjusted for Control
     **The coupling rate is 0.475 under these conditions.

• rhGNE: 1 µg
• Coupling Phosphatase 4: 0.1 µg
• ATP: 0.2 mM
• N-Acetyl-D-mannosamine: 1 mM