Recombinant Human Fucosyltransferase 7/FUT7 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: 100 mM Tris, 10 mM MnCl₂, 5 mM CaCl₂, pH 7.5
• Recombinant Human Fucosyltransferase 7/FUT7 (rhFUT7) (Catalog # 6409-GT)
• Donor Substrate: GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
• Acceptor Substrate: Fetuin, from Fetal Bovine Serum (Sigma, Catalog # F3385), 50 mg/mL in deionized water
• Glycosyltransferase Activity Kit (Catalog # EA001)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute Coupling Phosphatase 1 to 0.1 mg/mL in Assay Buffer.
2. Prepare reaction mixture by combining 30 µL of 1.6 mM GDP-Fucose, 120 µL of 50 mg/mL Fetuin, 24 µL of 0.1 mg/mL Coupling Phosphatase 1 and 246 µL of Assay Buffer. This volume is sufficient to assay 10 wells.
3. Dilute rhFUT7 to 5 µg/mL in Assay Buffer.
4. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 μM stock.
5. Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
7. Load 15 µL of the 5 µg/mL rhFUT7 into the plate. Include a Control containing 15 µL of Assay Buffer.
8. Add 35 µL of reaction mixture (step 2) to the wells, excluding the standard curve and curve blank.
9. Cover the plate with a plate sealer and incubate at 37 °C for 30 minutes.
10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
11. Add 100 µL of deionized water to all wells. Mix briefly.
12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
13. Read plate at 620 nm (absorbance) in endpoint mode.
    Calculate specific activity:

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

• rhFUT7: 0.075 µg
• Coupling Phosphatase 1: 0.2 µg
• Fetuin: 500 µg
• GDP-Fucose: 4000 pmol