Recombinant Human B3GNT4 Protein, CF

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

Recombinant Human B3GNT4 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer N-acetylglucosamine from UDP-GlcNAc to beta -lactose. The specific activity is >10 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Beta-1,3-N-Acetylglucosaminyltransferase 4/B3GNT4 protein
  Ala56-Arg378, with a C-terminal 6-His tag
• Accession #: Q9C0J1
• N-terminal Sequence: Ala56
• Protein/Peptide Type: Recombinant Enzymes
• Gene: B3GNT4
• 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: 37 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: 40-55 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 Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 25 mM Tris, 10 mM CaCl₂, 10 mM MnCl₂ (supplied in kit), pH 7.5
  • Recombinant Human beta -1,3-N-Acetylglucosaminyltransferase 4/B3GNT4 (rhB3GNT4) (Catalog # 7696‑GT)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
  • beta -Lactose (Sigma, Catalog # L3750), 250 mM stock in deionized water
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • 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 reaction mixture containing 80 mM beta -Lactose, 2 mM UDP-GlcNAc, and 4 µg/mL Coupling Phosphatase I in Assay Buffer.
  4. Dilute rhB3GNT4 to 12 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 the 12 µg/mL rhB3GNT4 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 with parafilm or a plate sealer and incubate at 37 ºC for 2 hours.
  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:
  • rhB3GNT4: 0.3 µg
  • Coupling Phosphatase I: 0.1 µg
  • beta -Lactose: 40 mM
  • UDP-GlcNAc: 1.0 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

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

Alternate Names for Recombinant Human B3GNT4 Protein, CF

• B3GNT4
• B3GN-T4
• beta-1,3-Gn-T4
• Beta-1,3-N-acetylglucosaminyltransferase 4
• beta-1,3-N-acetylglucosaminyltransferase bGn-T4
• beta3Gn-T4
• BGnT-4
• EC 2.4.1.-
• UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 4

Background

beta 1,3-Linked GlcNAc residues are present in the backbone of various biologically important glycans which are involved in many essential biological functions such as keratan sulfate synthesis in corneal tissue (1). The addition of such residues are catalyzed by a family of beta 1,3-N-acetylglucosaminyltransferases, that includes at least eight members (15). All of them are type II Golgi resident transmembrane proteins and have high homology to the beta 1,3-galactosyltransferase family. beta 1,3-N-acetylglucosaminyltransferase 4 or beta 3GNT4 is involved in polylactosamine synthesis and is mainly expressed in brain tissues such as whole brain, hippocampus, amygdala, cerebellum and caudate nucleus (6). The enzymatic activity of the recombinant protein was determined using a phosphatase coupled assay (7).

1. Seko, A. and Yamashita, K. (2005) Glycobiology 15:943.
2. Kataoka, K. and Huh, N.H. (2002) Biochem. Biophys. Res. Commun. 294:843.
3. Iwai,T. et al. (2002) J. Biol. Chem. 277:12802.
4. Togayachi, A. et al. (2001) J. Biol. Chem. 276:22032.
5. Sasaki, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94:14294.
6. Shiraishi, N. et al. (2001) J. Biol. Chem. 276:3498.
7. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: B3GNT4
• Uniprot:
  • Q9C0J1(Human)