Recombinant Human Fucosyltransferase 5/FUT5 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: 25 mM Tris, 5 mM MnCl₂ (supplied in kit), pH 7.5
• Recombinant Human Fucosyltransferase 5/FUT5 FUT5 (rhFUT5) (Catalog # 4949-GT)
• Donor Substrate: GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
• Acceptor Substrate: Lactosamine (V-Labs, Catalog # GN204), 50 mM 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 GDP-Fucose to 240 μM in Assay Buffer.
2. Dilute Lactosamine to 2.4 mM in Assay Buffer.
3. Dilute Coupling Phosphatase 1 to 12 μg/mL in Assay Buffer.
4. Prepare reaction mixture by combining equal volumes of diluted GDP-Fucose, Lactosamine, and Coupling Phosphatase 1.
5. Dilute rhFUT5 to 20 µg/mL in Assay Buffer.
6. 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.
7. 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.
8. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
9. Load 25 µL of the 20 µg/mL rhFUT5 into the plate. Include a Blank Control containing 25 µL of Assay Buffer.
10. Add 25 µL of reaction mixture (step 4) to the wells, excluding the standard curve and curve blank.
11. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells. Tap to mix briefly.
13. Add 100 µL of deionized water to all wells. Tap to 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 or 4-parameter fitting and adjusted for Blank Control.

• rhFUT5: 0.5 µg
• Coupling Phosphatase 1: 0.1 µg
• Lactosamine: 400 µM
• GDP-Fucose: 40 µM