Recombinant H. pylori Fucosyltransferase Protein, CF

• Glycosyltransferase Activity Kit (Catalog # EA001)
• Assay Buffer: 25 mM Tris, 10 mM CaCl₂, pH 7.0
• Recombinant H. pylori Fucosyltransferase (rHp FUT) (Catalog # 11072-GT)
• Lactosamine (Dextra Laboratories, Catalog # GN204), 50 mM stock in deionized water
• GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  

1. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 μL of the 1 mM Phosphate Standard to 360 μL of Assay Buffer for a 100 μM stock. This is the first point of the standard curve.
2. Complete the standard curve by performing six one-half serial dilutions of the 100 μM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
3. Prepare Reaction Mixture containing 2.4 mM Lactosamine, 0.240 mM GDP-Fucose, and 4 μg/mL Coupling Phosphatase 1 (provided in kit) in Assay Buffer.
4. Dilute rHp FUT to 1 μg/mL in Assay Buffer.
5. Load 50 μL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
6. Load 25 μL of 1 μg/mL of rHp FUT into the plate. Include a Control containing 25 μL of Assay Buffer.
7. Start the reactions by adding 25 μL of Reaction Mixture to the wells, excluding the standard curve.
8. Incubate sealed plate at 37 °C for 20 minutes.
9. Add 30 μL of the Malachite Green Reagent A to all wells. Mix briefly.
10. Add 100 μL of deionized water to all wells.
11. Add 30 μL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
12. Read plate at 620 nm (absorbance) in endpoint mode.
13. Calculate specific activity:
    
    

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

• rHp FUT: 0.025 µg
• Coupling Phosphatase 1: 0.1 μg
• Lactosamine: 1.2 mM
• GDP-Fucose: 0.12 mM