Recombinant Human Glucokinase/GCK Protein, CF

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

• Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MgCl₂, 10 mM CaCl₂, pH 7.0
• Recombinant Human Glucokinase/GCK (rhGCK) (Catalog # 7840-GK)
• Acceptor Substrate: Glucose (Sigma, Catalog # G5767), 2 M 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. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit to 100 µM by adding 40 µL of standard to 360 µL of Assay Buffer. This is the first point of the standard curve.
2. Perform six additional one‑half serial dilutions in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
3. Dilute Coupling Phosphatase 4 to 10 µg/mL in Assay Buffer.
4. Dilute rhGCK to 10 µg/mL in Assay Buffer.
5. Prepare a substrate mixture containing 0.5 mM ATP and 250 mM glucose in Assay Buffer.
6. Load 50 µL of each point of the standard curve to empty wells.
7. Load 20 µL dilute rhGCK to wells. Include Control containing 20 µL Assay Buffer.
8. Load 10 µL dilute Coupling Enzyme to wells used, excluding the standard curve.
9. Start the reaction by adding 20 µL substrate mixture to wells used, excluding the standard curve.
10. Cover the plate with a plate sealer and incubate at room temperature for 10 minutes.
11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
12. Add 100 µL of deionized water to all wells. Mix briefly.
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.
     **Under these conditions, the coupling rate is 0.475 (4).

• rhGCK: 0.2 µg
• Coupling Phosphatase 4: 0.1 µg
• ATP: 0.2 mM (10 nmol)
• Glucose: 100 mM (5000 nmol)