Recombinant Human GCNT1 Protein, CF

R&D Systems | Catalog # 7248-GT

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human GCNT1 Protein (7248-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

NS0

Accession Number

NP_001481

Applications

Enzyme Activity
View all Glucosaminyl (N-acetyl) Transferase 1/GCNT1 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Glucosaminyl (N-acetyl) Transferase 1/GCNT1 protein
Arg33-His428, with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

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

N-terminal Sequence Analysis

Arg33

Predicted Molecular Mass

47 kDa

SDS-PAGE

50-57 kDa, reducing conditions

Activity

Measured by its ability to transfer GlcNAc from UDP-GlcNAc to B1-3 galactosyl-N-acetyl galactosamine.
The specific activity is >350 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

7248-GT
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
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: Glucosaminyl (N-acetyl) Transferase 1/GCNT1

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. Glucosaminyl (N-acetyl) Transferase 1 (GCNT1) 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, 2). 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 (3, 4). The expression of GCNT1 was found to be associated with the progression of various types of cancer (5, 6, 7). The enzymatic activity of the recombinant GCNT1 is measured using a phosphatase-coupled method (8).

References

  1. Bierhuizen, M.F. (1993) Genes & Development 7:468.
  2. Yeh, J.C. et al. (1999) J. Biol. Chem. 274:3215.
  3. Hemmerich, S. et al. (1995) J. Biol. Chem. 270:12035.
  4. Wilkins, P.P. et al. (1996) J. Biol. Chem. 270:18732.
  5. Shimodaira, K. et al. (1997) Cancer Res. 57: 5201.
  6. Hatakyama, S. et al. (2010) Int. J. Cancer 127:1052.
  7. St Hill, C.A. et al. (2009) BMC Cancer 9:79.
  8. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Long Name

Glucosaminyl (N-acetyl) Transferase 1 Core 2

Alternate Names

C2GNT-L, C2GNT1, Core 2 GNT, G6NT, NACGT2

Entrez Gene IDs

2650 (Human); 14537 (Mouse); 64043 (Rat)

Gene Symbol

GCNT1

UniProt

NP_001481

Additional Glucosaminyl (N-acetyl) Transferase 1/GCNT1 Products

Product Documents for Recombinant Human GCNT1 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human GCNT1 Protein, CF

For research use only

Related Research Areas

Glycobiology-related Enzymes

Citations for Recombinant Human GCNT1 Protein, CF

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Protocols

View specific protocols for Recombinant Human GCNT1 Protein, CF (7248-GT):

Materials
  • Assay Buffer: 0.1 M MES, 10 mM DTT, 5 mM CaCl2, pH 6.0
  • Recombinant Human Glucosaminyl (N-acetyl) Transferase 1/GCNT1 (rhGCNT1) (Catalog # 7248-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
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 96-well Clear Plate (Catalog # DY990)
  • 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 a reaction mixture containing 0.4 mM UDP-Glc-NAc, 1 mM beta 1-3 Galactosyl-N-acetyl galactosamine and 4 μg/mL Coupling Phosphatase I in Assay Buffer.
  4. Dilute rhGCNT1 to 5 ng/µL 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 5 ng/µL rhGCNT1 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 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 incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. 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:

  • rhGCNT1: 0.125 µg
  • Coupling Phosphatase I: 0.1 µg
  • beta 1-3 Galactosyl-N-acetyl galactosamine: 0.5 mM
  • UDP-GlcNAc: 0.2 mM

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