Recombinant Human B4GalT1 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human B4GalT1 Protein, CF Summary

Details of Functionality
Measured by its ability to transfer galactose from UDP-galactose to N-Acetyl-alpha -D-glucosamine. The specific activity is >2,000 pmol/min/μg, as measured under the described conditions. 
Source
Mouse myeloma cell line, NS0-derived human beta-1,4-Galactosyltransferase 1/B4GalT1 protein
Gly44-Ser398, with an N-terminal 6-His tag
Accession #
N-terminal Sequence
His
Protein/Peptide Type
Recombinant Enzymes
Gene
B4GALT1
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
40 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
45-55 kDa
Publications
Read Publication using
3609-GT in the following applications:

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 under reducing conditions and visualized by silver stain
Assay Procedure
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MgCl2, 10 mM MnCl2 (supplied in kit), pH 7.5
  • Recombinant Human beta -1,4‑Galactosyltransferase 1/B4GalT1 (rhB4GalT1) (Catalog # 3609-GT)
  • Donor Substrate: UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
  • Acceptor Substrate: N-Acetyl-alpha -D-glucosamine (GlcNAc) (EMD, Catalog # 1079), 1 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 UDP-Galactose to 0.5 mM in Assay Buffer.
  2. Dilute Coupling Phosphatase 1 to 20 µg/mL in Assay Buffer.
  3. Dilute GlcNAc to 50 mM in Assay Buffer.
  4. Prepare Reaction Mixture by combining equal volumes of 0.5 mM UDP-GalNAc, 20 µg/mL Coupling Phosphatase 1, and 50 mM GlcNAc.
  5. Dilute rhB4GalT1 to 0.75 µg/mL in Assay Buffer.
  6. 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.
  7. 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.
  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 20 µL of the 0.75 µg/mL rhB4GalT1 into the plate. Include a Control containing 20 µL of Assay Buffer.
  10. Start the reaction by adding 30 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
  11. Seal plate and incubate at room temperature for 15 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 Well:
  • rhB4GalT1:  0.015 µg
  • Coupling Phosphatase 1: 0.2 µg
  • UDP-Galactose: 5 nmol
  • GlcNAc: 500 nmol

Notes

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

Alternate Names for Recombinant Human B4GalT1 Protein, CF

  • 1,4- galactosyltransferase, polypeptide 1
  • B4GalT1
  • B4GAL-T1
  • beta-1,4-galactosyltransferase 1
  • Beta-1,4-GalTase 1
  • beta4Gal-T1
  • betaGlcNAc beta
  • CDG2D
  • EC 2.4.1
  • GGTB2
  • GGTB2DKFZp686N19253
  • glycoprotein-4-beta-galactosyltransferase 2
  • GT1
  • GTB
  • lactose synthase
  • MGC50983
  • UDP-Gal
  • UDP-Gal:beta-GlcNAc beta-1,4-galactosyltransferase 1
  • UDP-galactose:beta-N-acetylglucosamine beta-1,4-galactosyltransferase 1

Background

beta 4GalT1 is one of seven beta 1,4 galactosyltransferases that transfer galactose in a beta 1,4 linkage to acceptor sugars including GlcNAc, and 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). beta 4GalT1 is unique among the seven enzymes because it can be expressed either as membrane associated form or secreted form (2). The secreted form is restricted to lactating mammary tissues where the enzyme forms a heterodimer with alpha -lactalbumin to catalyze the synthesis of lactose (3). The membrane form can reside either in the Golgi apparatus, where it adds galactose to N-acetylglucosamine residues, or on cell surface, where it functions as a recognition molecule during a variety of cell to cell and cell to matrix interactions, by binding to specific oligosaccharide ligands on opposing cells or in the extracellular matrix (4). The two enzymatic forms result from alternate transcription initiation sites and post-translational processing (5). Defects in beta 4GalT1 are the cause of congenital disorder of glycosylation type 2D (CDG2D) (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. Yamaguchi, N and Fukuda, M.N. (1995) J. Biol. Chem. 270:12170.
  3. Appert, H.E. et al. (1986) Biochem. Biophys. Res. Commun. 138:224.
  4. Lopez, L.C. et al. (1991) J. Biol. Chem. 266:15984.
  5. Mengle-Gaw, L. et al. (1991) Biochem. Biophys. Res. Commun. 176:1269.
  6. Hansske, B. et al. (2002) J. Clin. Invest. 109:725.
  7. Wu, Z.L. et al. (2011) Glycobiology 21:727.

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Publications for beta-1,4-Galactosyltransferase 1/B4GalT1 (3609-GT)(1)

We have publications tested in 1 confirmed species: Mouse.

We have publications tested in 1 application: Bioassay.


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Bioinformatics

Gene Symbol B4GALT1
Entrez
Uniprot