Recombinant Human Guanylate Kinase Protein, CF

• Universal Kinase Activity Kit (Catalog # EA004)
• 10X Assay Buffer (supplied in kit):  250 mM HEPES, 1.5 M NaCl, 100 mM MgCl₂, 100 mM CaCl₂, pH 7.0
• Recombinant Human Guanylate Kinase (rhGUK-1) (Catalog # 9267-GU)
• Guanosine 5'-monophosphate (GMP) (Sigma, Catalog # G8377), 10 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 stock 10 fold with deionized water.
2. Dilute 1 mM Phosphate Standard 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 point of the standard curve.
3. Continue 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 nmoles per well.
4. Prepare a reaction mixture containing 0.2 mM ATP (supplied in kit) and 0.2 mM GMP in 1X Assay Buffer.
5. Dilute rhGUK-1 to 0.0667 µg/mL 1X Assay Buffer.
6. Load 50 µL of each dilution of the standard curve into a plate.  Include a curve blank containing 50 μL of 1X Assay Buffer.
7. Load 15 µL of the 0.0667 µg/mL rhGUK-1 into empty wells of the same plate as the curve. Include a Control containing 15 µL of 1X Assay Buffer.
8. Add 25 µL of reaction mixture to all wells, excluding standard curve.
9. Seal plate and incubate at room temperature for 30 minutes.
10. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL 1X Assay Buffer.
11. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to wells containing enzyme and control, excluding the standard curve.
12. Seal plate and incubate at room temperature for 5 minutes.
13. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix briefly.
14. Add 100 µL of deionized water 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 or 4-parameter fitting and adjusted for Control.
**Incubation time is 35 minutes according to the above protocol.
***Both ADP and GDP can be digested by Coupling Phosphatase 4 and release one unit of phosphate.

Per Reaction:

• rhGUK-1: 0.001 µg
• Coupling Phosphatase 4: 0.1 µg
• ATP: 0.1 mM
• GMP: 0.1 mM