Recombinant Human A4GNT Protein, CF

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

Recombinant Human A4GNT Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer GlcNAc from UDP-GlcNAc to galactose. The specific activity is >400 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Alpha-1,4-N-Acetylglucosaminyltransferase 4/A4GNT protein
  Leu25-Lys340, with C-terminal 6-His tag
• Accession #: Q9UNA3
• N-terminal Sequence: Leu25
• Protein/Peptide Type: Recombinant Enzymes
• Gene: A4GNT
• 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: 38 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: 39-49 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 and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl₂, pH 7.5
  • MnCl₂ (supplied in kit): 100 mM
  • Recombinant Human alpha -1,4-N-Acetylglucosaminyltransferase 4/A4GNT (rhA4GNT) (Catalog # 8960-GT)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
  • D-(+)-Galactose (Sigma, Catalog # G0625), 750 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer containing 10 mM MnCl₂ by combining 10X stocks and diluting 10-fold with deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Prepare reaction mixture containing 5 mM UDP-GlcNAc, 200 mM D-(+)-Galactose, and 4 µg/mL Coupling Phosphatase 1 in 1X Assay Buffer.
  5. Dilute rhA4GNT to 4 ng/µL in 1X Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  7. Load 25 µL of 4 ng/µL rhA4GNT into empty wells of the same plate as the curve. Include a Control containing 25 μL of 1X Assay Buffer.
  8. Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
  9. Seal plate and incubate at 37°C for 20 minutes.
  10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
  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:
  • rhA4GNT: 0.1 µg
  • Coupling Phosphatase 1: 0.1 µg
  • UDP-GlcNac: 2.5 mM
  • D-(+)-Galactose: 100 mM

Notes

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

Alternate Names for Recombinant Human A4GNT Protein, CF

• A4GNT
• alpha-1,4-N-acetylglucosaminyltransferase
• alpha4GnT

Background

Alpha-1,4-N-acetylglucosaminyltransferase A4GNT is a type II Golgi resident membrane glycosyltransferase, but shows no significant sequence similarity with any other glycosyltransferases. It catalyzes the transfer of N-acetylglucosamine (GlcNAc) to core 2 branched O-glycans and forms a unique glycan,
GlcNAc alpha 1-->4Gal beta -->R on mucin (1). Alpha-1,4-GlcNAc-capped mucin-type O-glycan inhibits cholesterol alpha -glucosyltransferase from Helicobacter pylori (Hp) and suppresses Hp growth (2). Hp infection is widespread and thought to be the causes of the development of gastric lesions including gastritis, intestinal metaplasia, and gastric carcinoma (3). The enzymatic activity of the recombinant protein was determined using a phosphatase-coupled assay (4).

1. Nakayama, J. et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8991
2. Lee, H. et al. (2008) Glycobiology 18:549.
3. Ferreira, B. et al. (2006) J. Histochem. Cytochem. 54: 585.
4. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: A4GNT
• Uniprot:
  • Q9UNA3()