Recombinant Human C2GNT3/GCNT4 His-tag Protein

• Glycosyltransferase Activity Kit (Catalog # EA001)
• Assay Buffer:  0.1 M MES, 10 mM DTT, 5 mM CaCl₂, pH 6.0
• Recombinant Human C2GNT3/GCNT4 (rhGCNT4) (Catalog # 11162-GT)
• UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
• beta -1,3 Galactosyl-N-acetyl galactosamine (V-labs, Catalog # GN213), 20 mM stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare a standard curve from the 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.
2. Continue standard curve by performing six additional one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
3. Prepare a Reaction Mixture containing 0.4 mM UDP-GlcNAc, 1 mM beta -1,3 Galactosyl-N-acetyl galactosamine, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
4. Dilute rhGCNT4 to 20 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 20 µg/mL rhGCNT4 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
7. Start the reactions by adding 25 µL of Reaction Mixture to all wells, excluding the standard curve and curve blank.  
8. Seal plate and incubate at 37 °C for 20 minutes.
9. Add 30 µL of the Malachite Green Reagent A to all wells used, including standard curve. Mix briefly.
10. Add 100 µL of deionized water to all wells used, including standard curve. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells used, including standard curve. Mix briefly.
12. Seal plate and incubate at room temperature for 20 minutes.
13. Read plate at 620 nm (absorbance) in endpoint mode.
14. Calculate specific activity:
    
    

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

• rhGCNT4: 0.5 µg
• Coupling Phosphatase 1: 0.1 µg
• beta -1,3 Galactosyl-N-acetyl galactosamine: 0.5 mM
• UDP-GlcNAc: 0.2 mM
  
  
  
  
  
  
  
  
  
  
  
  

Scientific Data

Enzyme Activity View Larger

GCNT4 (Catalog # 11162-GT) recognizes core-1 O-glycan and convert it to core-2 type O-glycan.

SDS-PAGE View Larger

1 μg/lane of Recombinant Human GCNT4 His-tag (Catalog # 11162-GT ) was resolved with SDS-PAGE under reducing conditions and visualized by silver staining, showing a band at 50-66 kDa.