Recombinant Human B3GNT4 Protein, CF

• Assay Buffer: 25 mM Tris, 10 mM CaCl₂, 10 mM MnCl₂ (supplied in kit), pH 7.5
• Recombinant Human beta -1,3-N-Acetylglucosaminyltransferase 4/B3GNT4 (rhB3GNT4) (Catalog # 7696‑GT)
• UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
• beta -Lactose (Sigma, Catalog # L3750), 250 mM 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 by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
2. Prepare 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 80 mM beta -Lactose, 2 mM UDP-GlcNAc, and 4 µg/mL Coupling Phosphatase I in Assay Buffer.
4. Dilute rhB3GNT4 to 12 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 12 µg/mL rhB3GNT4 into the plate. Include a Control containing 25 µL of Assay Buffer.
7. Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
8. Seal the plate with parafilm or a plate sealer and incubate at 37 ºC for 2 hours.
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 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:

• rhB3GNT4:  0.3 µg
• Coupling Phosphatase I: 0.1 µg
• beta -Lactose: 40 mM
• UDP-GlcNAc: 1.0 mM