Recombinant Human GCNT2 His-tag Protein, CF

GCNT2 (Catalog # 10847-GT) recognizes polylactosamine structure and can tolerate core-6 fucose and its Cy5 derivatives.

1 μg/lane of Recombinant Human GCNT2 His-tag (Catalog # 10847-GT) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing bands at 53-63 kDa.

Lane 1 contained substrate glycan extended G2 (GL303). In the presence of rhGCNT2, the glycan was modified, and a mobility shift was observed.

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

Recombinant Human GCNT2 His-tag Protein, CF Summary

• Additional Information: Glucosaminyl (N-acetyl) Transferase 2
• Details of Functionality: Measured by its ability to transfer GlcNAc from UDP-GlcNAc to Cy5-labeled Extended G2.
  
  0.25 μg of Recombinant Human GCNT2 will convert >50% of its substrate to product, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Glucosaminyl (N-acetyl) Transferase 2/GCNT2 protein
  Asn26-Phe400, with a C-terminal 6-His
• Accession #: NP_001482.1
• N-terminal Sequence: Asn26
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 43.8 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: 53-67 kDa, under 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 and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, 1 mM DTT, pH 6.5
  • Recombinant Human GCNT2 (rhGCNT2) (Catalog # 10847-GT)
  • Cy5-labeled Extended G2 (Catalog # GL303)
  • Recombinant Human B4GalT1 (rhB4GALT1) (Catalog # 3609-GT)
  • UDP-Gal (Sigma, Catalog # U4500), 10 mM stock in Deionized Water
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol/50% Deionized Water
    15% SDS-PAGE Gel
  • Reducing SDS-PAGE gel loading buffer
  • Fluorescent imager
  1. Dilute rhGCNT2 to 25 µg/mL in Assay Buffer.
  2. Prepare a Master Mix containing 0.2 µM Cy5-labeled Extended G2, 1 mM UDP-GlcNAc, 1 mM UDP-Gal and 50 µg/mL rhB4GALT1 in Assay Buffer.
  3. Prepare reaction(s) by combining 10 µL of 25 µg/mL rhGCNT2 and 10 µL of Master Mix. Prepare a negative control by combining 10 µL of Assay Buffer and 10 µL of Master Mix.
  4. Incubate the reaction(s) and control at 37 °C for 90 minutes.
  5. Add 7 µL of reducing SDS-PAGE gel loading buffer to each reaction and control. Mix.
  6. Load 13.5 µL of each reaction and control per lane on a 15% SDS-PAGE gel. Run dye front down 80% of the length of the gel (minimum).
  7. Visualize the gel with a fluorescent imager.
  8. Calculate percent conversion of the substrate to product.
  Per Reaction:
  • rhGCNT2: 0.25 µg
  • Cy5-labeled Extended G2: 2 pmol
  • UDP-Gal: 0.5 mM
  • UDP-GlcNAc: 0.5 mM
  • rhB4GALT1: 0.5 µg

Notes

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

Alternate Names for Recombinant Human GCNT2 His-tag Protein, CF

• bA360O19.2
• bA421M1.1
• beta-1,6-N-acetylglucosaminyltransferase 2
• cataract, congenital
• CCAT
• CTRCT13
• EC 2.4.1.150
• GCNT2
• GCNT2C
• GCNT5
• glucosaminyl (N-acetyl) transferase 2, I-branching enzyme (I blood group)
• glucosaminyl (N-acetyl) transferase 2, I-branching enzyme (Ii blood group)
• glucosaminyl (N-acetyl) transferase 2, I-branching enzyme
• I beta-1,6-N-acetylglucosaminyltransferase
• I-branching beta-1,6-acetylglucosaminyltransferase
• I-branching enzyme
• IGNTglucosaminyl (N-acetyl) transferase 5
• Ii blood group
• II
• MGC163396
• N-acetylglucosaminyltransferase
• N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase
• N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase
• NACGT1
• NAGCT1
• ULG3

Background

N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase (GCNT2) is a key branching enzyme that converts linear into branched poly-N-acetyllactosaminoglycans. It is responsible for the formation of the blood group I antigens during embryonic development and is closely associated with the development and maturation of erythroid cells (1, 2, 3). GCNT2 is a Golgi resident type II membrane protein with a typical domain structure of glycosyltransferases. GCNT2 has been found to be downregulated in melanomas, which leads to the loss of N-linked I-branched glycans and the synthesis of poly-N-acetyllactosamine (i-linear) glycans (4). In addition, GCNT2 has been found to play a role in breast cancer and lung cancer and knockdown of GCNT2 expression decreased cell migration and invasion in vitro (5). The activity of recombinant GCNT2 is demonstrated in an electrophoretic gel mobility shift assay using a fluorophore-labeled glycan as the substrate (6).

1. Marti, F.A. et al. (1995) Glycobiology 5:417.
2. Bierhuizen, M.F.A. et al. (1993) Genes Dev. 7:468.
3. Inaba, N. et al. (2003) blood 101:2870.
4. Sweeney, Jenna Geddes et al. (2018) Nature communications 9:3368.
5. Haijun, Zhang et al. (2011) Cancer Res. 71:4846.
6. Wu, Z.L. et al (2020) Glycobiology 30:970.

Bioinformatics

• Uniprot:
  • NP_001482.1()