Recombinant Human Hexokinase 1 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.

• Universal Kinase Activity Kit (Catalog # EA004)
• 10X Assay Buffer (supplied in kit): 250 mM HEPES, 1500 mM NaCl, 100 mM MgCl₂, 100 mM CaCl₂, pH 7.0
• Recombinant Human Hexokinase 1 (rhHK-1) (Catalog # 8178-HK)
• Glucose (Sigma, Catalog # G5767), 100 mM stock in deionized water
• 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 with 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 (this is the first standard curve dilution).
3. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer.  The standard curve has a range of 0.078 to 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 0.5 mM ATP and 25 mM Glucose in 1X Assay Buffer.
6. Dilute rhHK-1 to 7.5 ng/µL in 1X Assay Buffer.
7. Load 20 µL of the 7.5  ng/µL rhHK-1 into the plate in triplicate. Include a Control containing 20 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 20 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.

• rhHK-1: 0.15 µg
• Coupling Phosphatase 4: 0.1 µg
• ATP: 0.2 mM
• Glucose: 10 mM