Recombinant Human GCNT1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human GCNT1 Protein, CF Summary

• Details of Functionality: 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.
• Source: Mouse myeloma cell line, NS0-derived human Glucosaminyl (N-acetyl) Transferase 1/GCNT1 protein
  Arg33-His428, with a C-terminal 6-His tag
• Accession #: NP_001481
• N-terminal Sequence: Arg33
• Protein/Peptide Type: Recombinant Enzymes
• Gene: GCNT1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 47 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 50-57 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human GCNT1 Protein, CF

• beta-1,3-galactosyl-O-glycosyl-glycoproteinbeta-1,6-N-acetylglucosaminyltransferase
• beta-1,6-N-acetylglucosaminyltransferase
• C2GNT1
• C2GNTbeta-1,3-galactosyl-O-glycosyl-glycoproteinbeta-1,6-N--acetylglucosaminyltransferase
• C2GNT-L
• core 2 beta1,6 N-acetylglucosaminyltransferase-I
• core 2 beta-1,6-N-acetylglucosaminyltransferase I
• core 2 branching enzyme
• Core 2 GNT
• Core 2-branching enzyme
• core2-GlcNAc-transferase
• EC 2.4.1.102
• G6NT
• GCNT1
• glucosaminyl (N-acetyl) transferase 1, core 2
• glucosaminyl (N-acetyl) transferase 1, core 2(beta-1,6-N-acetylglucosaminyltransferase)
• MGC126335
• MGC126336
• NACGT2
• NAGCT2

Background

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 Le^x 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).

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.

Publications for Glucosaminyl (N-acetyl) Transferase 1/GCNT1 (7248-GT)(4)

Publications using 7248-GT

• ZL Wu, JM Ertelt Fluorescent glycan fingerprinting of SARS2 spike proteins Scientific Reports, 2021-10-14;11(1):20428. 2021-10-14 [PMID: 34650101] (Bioassay, Human)
• B Trastoy, A Naegeli, I Anso, J Sjögren, ME Guerin Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila Nat Commun, 2020-09-24;11(1):4844. 2020-09-24 [PMID: 32973204] (Bioassay, E. coli)
• Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R Imaging specific cellular glycan structures using glycosyltransferases via click chemistry. Glycobiology, 2018-02-01;0(0):. 2018-02-01 [PMID: 29186441] (Click Chemistry, Human, Mouse)
• Kojima Y, Yoneyama T, Hatakeyama S, Mikami J, Sato T, Mori K, Hashimoto Y, Koie T, Ohyama C, Fukuda M, Tobisawa Y Detection of Core2 beta-1,6-N-Acetylglucosaminyltransferase in Post-Digital Rectal Examination Urine Is a Reliable Indicator for Extracapsular Extension of Prostate Cancer. PLoS ONE, 2015-09-21;10(9):e0138520. 2015-09-21 [PMID: 26390303] (Western Blot, Human)

Bioinformatics

• Gene Symbol: GCNT1
• Uniprot:
  • NP_001481()