Recombinant Human C2GNT3/GCNT4 His-tag Protein

R&D Systems | Catalog # 11162-GT

Glucosaminyl (N-acetyl) Transferase 4 Core 2
R&D Systems
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Key Product Details

  • R&D Systems CHO-derived Recombinant Human C2GNT3/GCNT4 His-tag Protein (11162-GT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

Q9P109.1

Applications

Enzyme Activity
View all C2GNT3/GCNT4 Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human C2GNT3/GCNT4 protein
Arg35-Ser453, with C-terminal 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Arg35

Predicted Molecular Mass

49.7 kDa

SDS-PAGE

50-66 kDa, under reducing conditions.

Activity

Measured by its ability to transfer GlcNAc from UDP-GlcNAc to B1-3 galactosyl-N-acetyl galactosamine.
The specific activity is >75 pmol/min/μg, as measured under the described conditions.

Scientific Data Images for Recombinant Human C2GNT3/GCNT4 His-tag Protein

Recombinant Human C2GNT3/GCNT4 His-tag Protein/GCNT4 Enzyme Activity Diagram

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

Recombinant Human C2GNT3/GCNT4 His-tag Protein SDS-PAGE

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.

Formulation, Preparation, and Storage

11162-GT
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: C2GNT3/GCNT4

Mucin-type O-glycans are initiated with an O-GalNAc attachment to a serine or threonine in a polypeptide. The O-GalNAc residues are subsequently extended by various glycosyltransferases resulting in different types of O-glycans. Most O-glycans contain the core 1 structure, Gal beta 1-3GalNAc. GCNT4 is a member of the glucosaminyl (N-acetyl) transferase family and it converts the core 1 O-glycan to core 2 O-glycan, Gal beta 1-3(GlcNAc beta 1-6)GalNAc, via the addition of a GlcNAc residue (1). Various ligand carbohydrates can be formed from core 2 branched oligosaccharides. For example, sialyl Lex in mucin‑type glycoproteins of blood cells can be formed from core 2 branched oligosaccharides (2,3). GCNT4 does not have core 4 O-glycan or I-branching enzyme activity. GCNT4 has been shown to play a role in gastric cancers, where GCNT4 is downregulated and its overexpression prevents cancer cell growth (4, 5). The activity of the recombinant human GCNT-4 was determined using a phosphatase‑coupled glycosyltransferase assay (6).

References

  1. Schwientek, T. et al. (2000) J Biol Chem. 275:11106.
  2. Hemmerich, S. et al. (1995) J. Biol. Chem. 270:12035.
  3. Wilkins, P.P. et al. (1996) J. Biol. Chem. 270:18732.
  4. Sun, H. et al. (2020) Onco. Targets Ther. 25:8601.
  5. Hu, W. et al. (2021) Bioengineered 12:11634.
  6. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Long Name

Glucosaminyl (N-acetyl) Transferase 4 Core 2

Alternate Names

GCNT4, Gm279, Gm73

Entrez Gene IDs

51301 (Human); 218476 (Mouse); 310011 (Rat)

Gene Symbol

GCNT4

UniProt

Q9P109.1

Additional C2GNT3/GCNT4 Products

Product Documents for Recombinant Human C2GNT3/GCNT4 His-tag Protein

Certificate of Analysis

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Product Specific Notices for Recombinant Human C2GNT3/GCNT4 His-tag Protein

For research use only

Related Research Areas

Glycobiology-related Enzymes

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Protocols

View specific protocols for Recombinant Human C2GNT3/GCNT4 His-tag Protein (11162-GT):

Materials
  • 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:

     Specific Activity (pmol/min/µg) =

 Phosphate released* (nmol) x (1000 pmol/nmol)
Incubation time (min) x amount of enzyme (µg)

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

Per Reaction:
  • 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











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