Recombinant Human Fucosyltransferase 2/FUT2 Protein, CF

• Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl₂, 10 mM CaCl₂, pH 7.5
• Recombinant Human FUT2 (rhFUT2) (Catalog # 7770-GT)
• GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
• alpha -lactose (Sigma, Catalog # L2643), 0.3 M stock 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 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. Prepare reaction mixture containg 100 mM alpha -lactose, 200 µM GDP-Fucose, and 4 µg/mL Coupling Phosphatase I in Assay Buffer.
4. Dilute rhFUT2 to 1.0 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 the 1 µg/mL rhFUT2 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
7. Add 25 µL of reaction mixture (step 3) to the wells, excluding the standard curve and curve blank.
8. Cover the plate with a plate sealer and incubate 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. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and inubate 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:

• rhFUT-2: 0.025 µg
• Coupling Phosphatase I: 0.1 µg
• alpha -Lactose: 50 mM
• GDP-Fucose: 100 µM