Recombinant Human B4GalT2 Protein, CF

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

Recombinant Human B4GalT2 Protein, CF Summary

• Details of Functionality: Measured by its ability to transfer galactose from UDP-galactose to glucose. The specific activity is >80 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human beta-1,4-Galactosyltransferase 2/B4GalT2 protein
  Ser43-Gly372, with C-terminal 6-His tag
• Accession #: O60909
• N-terminal Sequence: Ser43
• Protein/Peptide Type: Recombinant Enzymes
• Gene: B4GALT2
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <0.10 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: 44-55 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -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: >90%, 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 MnCl₂, 10 mM CaCl₂, 65 mM NaCl, pH 7.5
  • Recombinant Human beta ‑1,4‑Galactosyltransferase 2/B4GalT2 (rh beta 4GalT2) (Catalog # 7530-GT)
  • UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
  • D-(+)-Glucose (Sigma, Catalog # G5767), 2 M 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 by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  2. Continue 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 5.0 nmol per well.
  3. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  4. Dilute rh beta 4GalT2 to 16 µg/mL.
  5. Prepare a Reaction Mixture composed of 0.4 M Glucose, 1 mM UDP-Galactose, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
  6. Load 25 µL of the 16 µg/mL rh beta 4GalT2 into the plate. Include a Control containing 25 µL of Assay Buffer.
  7. Start the reaction by adding 25 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
  8. Cover the plate with a plate sealer and incubate at room temperature 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 Well:
  • rh beta 4GalT2: 0.4 µg
  • Coupling Phosphatase 1: 0.1 µg
  • UDP-Galactose: 0.5 mM
  • Glucose: 200 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 B4GalT2 Protein, CF

• B4GalT2
• B4Gal-T2
• B4Gal-T3
• beta-1,4-Galactosyltransferase 2
• Beta-1,4-GalTase 2
• beta-4-GalT2
• beta4Gal-T2
• beta-N-acetylglucosaminyl-glycolipid beta-1,4-galactosyltransferase 2
• EC 2.4.1.-
• UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase 2
• UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 2
• UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 2
• UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 2
• UDP-galactose:beta-N-acetylglucosamine beta-1,4-galactosyltransferase 2

Background

There are seven beta -1,4-galactosyltransferases that transfer galactose in a beta -1,4 linkage to acceptor sugars including GlcNAc, Glc, and Xyl. By sequence similarity, the beta 4GalTs form four groups: beta 4GalT1 and beta 4GalT2, beta 4GalT3 and beta 4GalT4, beta 4GalT5 and beta 4GalT6, and beta 4GalT7 (1). All of these enzymes are expressed as type II membrane proteins in Golgi apparatus except beta 4GalT1, which can be expressed as a secreted form in lactating mammary tissues due to an alternative transcription initiation site (2, 3). beta 4GalT2 is responsible for the synthesis of complex-type N-linked oligosaccharides in many glycoproteins as well as the carbohydrate moieties of glycolipids (4). It can also produce lactose. Its substrate specificity is affected by alpha -lactalbumin, but it is not expressed in lactating mammary tissue (5). Recently, beta 4GalT2 has been shown to form a complex with GlcAT-P to synthesize HNK-1 carbohydrate in the nervous system (6). The activity of this enzyme has been measured with a phosphatase-coupled method (7).

1. Amado, M. et al. (1999) Biochim. Biophys. Acta. 1473:35.
2. Appert, H.E. et al. (1986) Biochem. Biophys. Res. Commun.138:224.
3. Mengle-Gaw,L. et al. (1991) Biochem. Biophys. Res.Commun. 176:1269.
4. Guo, S. et al. (2001) Glycobiology 11:813.
5. Almeida, R. et al. (1997) J. Biol. Chem. 272:31979.
6. Kouno, T. et al. (2011) J. Biol. Chem. 286:31337.
7. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Bioinformatics

• Gene Symbol: B4GALT2
• Uniprot:
  • O60909(Human)