Recombinant Human B3GNT2 Protein, CF

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

Recombinant Human B3GNT2 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer GlcNAc from UDP-GlcNAc to N-Acetyl-D-Lactosamine. The specific activity is > 50 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Beta-1,3-N-Acetylglucosaminyltransferase 2/B3GNT2 protein
  Lys29-Cys397, with N-terminal 6-His tag
• Accession #: Q9NY97
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: B3GNT2
• 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: 44 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: 63-90 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -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: 25 mM Tris, 10 mM CaCl₂, 10 mM MnCl₂ (supplied in kit), pH 7.5
  • Recombinant Human beta -1,3-N-acetylglucosaminyltransferase 2/B3GNT2 (rhB3GNT2) (Catalog # 3960-GT)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
  • N-acetyllactosamine (V-Labs, Catalog # GN204), 50 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. Dilute UDP-GlcNAc to 1.8 mM in Assay Buffer.
  4. Dilute N-acetyllactosamine to 6 mM in Assay Buffer.
  5. Dilute Coupling Phosphatase I to 6 ng/µL in Assay Buffer.
  6. Prepare reaction mixture by combining equal volumes of 1.8 mM UDP-GlcNAc, 6 mM N-acetyllactosamine, and 6 ng/µL Coupling Phosphatase I.
  7. Dilute rhB3GNT2 to 8 ng/µL in Assay Buffer.
  8. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  9. Load 25 µL of the 8 ng/µL rhB3GNT2 into the plate. Include a Control containing 25 µL of Assay Buffer.
  10. Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
  11. Seal the plate and incubate at 37 °C for 20 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  13. Add 100 µL of deionized water to all wells. Mix briefly.
  14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  15. Read plate at 620 nm (absorbance) in endpoint mode.
  16. 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:
  • rhB3GNT2: 0.2 µg
  • Coupling Phosphatase I: 0.05 µg
  • N-acetyllactosamine: 1 mM
  • UDP-GlcNAc: 0.3 mM

Notes

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

Alternate Names for Recombinant Human B3GNT2 Protein, CF

• B3Galt6
• B3GALT7
• b3Gal-T7
• B3GNT
• B3GNT1
• B3GN-T1
• B3GNT2
• B3GN-T2
• B3GNT-2
• Beta-1,3-galactosyltransferase 7
• Beta-1,3-GalTase 7
• Beta-1,3-Gn-T1
• Beta-1,3-Gn-T2
• Beta-1,3-N-acetylglucosaminyltransferase 1
• Beta-1,3-N-acetylglucosaminyltransferase 2
• beta-1,3-N-acetylglucosaminyltransferase bGnT-1
• beta-1,3-N-acetylglucosaminyltransferase bGnT-2
• Beta3GalT7
• Beta3Gal-T7
• BETA3GNT
• Beta3gnt1
• beta3Gn-T1
• Beta3Gn-T2
• Beta-3-Gx-T7
• BGnT-1
• BGNT2
• BGnT-2
• EC 2.4.1.-
• UDP-Gal:beta-GlcNAc beta-1,3-galactosyltransferase 7
• UDP-galactose:beta-N-acetylglucosamine beta-1,3-galactosyltransferase 7
• UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1
• UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2

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 (1-5). 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‑acetylglucosaminyltransferases 2 or beta 3GNT2 prefers the substrate lacto-N-neotetraose and is involved in the biosynthesis of poly‑N‑acetyllactosamine chains (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: B3GNT2
• Uniprot:
  • Q9NY97()