Recombinant Human B3GAT1 Protein, CF

R&D Systems | Catalog # 8560-GT

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Key Product Details

  • R&D Systems CHO-derived Recombinant Human B3GAT1 Protein (8560-GT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

CHO

Accession Number

Q9P2W7

Applications

Enzyme Activity
View all beta-1,3-Glucuronyltransferase 1/B3GAT1 Proteins and Enzymes »

Product Specifications

Source

Chinese Hamster Ovary cell line, CHO-derived human beta-1,3-Glucuronyltransferase 1/B3GAT1 protein
His25-Ile334, with N-terminal 6-His tag

Purity

>85%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<0.01 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

His

Predicted Molecular Mass

36 kDa

SDS-PAGE

50-59 kDa, reducing conditions

Activity

Measured by its ability to transfer GlcA from UDP-GlcA to lactose.
The specific activity is >750 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

8560-GT
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Background: beta-1,3-Glucuronyltransferase 1/B3GAT1

B3GAT1 is a key enzyme involved in human natural killer1 (HNK1) epitope synthesis. It adds a glucuronic residue to the terminal lactosamine residue (Gal beta 14GlcNAc) of a glycoprotein or glycolipid, which can be further sulfated to become the HNK1 epitope, a unique trisaccharide structure, HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc (1, 2). The enzyme activity was found to be enhanced in the presence of sphingomyelin and phosphatidylinositol (3). The HNK1 carbohydrate epitope is characteristically expressed on a series of cell adhesion molecules in addition to some glycolipids in the extracellular matrix and on the cell surface in the nervous system, where it is involved in cell-cell and cell-substratum interaction and recognition during the development of the nervous system (4). Like most known glycosyltransferases, B3GAT1 is a type II Golgi-resident transmembrane protein with a short N-terminal cytoplasmic domain and a single pass transmembrane domain followed by an enzymatic domain in the lumen of Golgi apparatus. The enzyme activity was assayed using a phosphatase-coupled method (5).

References

  1. Terayama, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94:6093.
  2. Shogo, O. et al. (1992) J. Biol. Chem. 267: 22711.
  3. Kakuda, S. et al. (2005) Glycobiology 2:203.
  4. Bollensen, E. and Schachner, M. (1987) Neurosci Lett. 82:77.
  5. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Long Name

UDP-GlcUA:glycoprotein beta-1,3-Glucuronyltransferase

Alternate Names

CD57, GLCATP, GLCUATP, HNK1, LEU7

Entrez Gene IDs

27087 (Human); 76898 (Mouse); 117108 (Rat)

Gene Symbol

B3GAT1

UniProt

Q9P2W7

Additional beta-1,3-Glucuronyltransferase 1/B3GAT1 Products

Product Documents for Recombinant Human B3GAT1 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human B3GAT1 Protein, CF

For research use only

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Protocols

View specific protocols for Recombinant Human B3GAT1 Protein, CF (8560-GT):

Materials
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM MnCl2, 5 mM CaCl2, pH 7.0
  • Recombinant Human beta -1,3-Glucuronyltransferase 1/B3GAT1 (rhB3GAT1) (Catalog # 8560-GT)
  • UDP-GlcA (Sigma, Catalog # U5625), 10 mM stock in deionized water.
  • alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • 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. This is the first point of the standard curve.
  2. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  3. Prepare reaction mixture containing 1.25 mM UDP-GlcA, 60 mM alpha -Lactose, and 8 ng/μL Coupling Phosphatase 1 in Assay Buffer.
  4. Dilute rhB3GAT1 to 2 ng/µL 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 2 ng/µL rhB3GAT1 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Add 25 µL of the reaction mixture to all wells, excluding the standard curve. 
  8. Seal plate and incubate at 37 °C 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 sealed plate 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:

  • rhB3GAT1: 0.050 µg
  • Coupling Phosphatase 1: 0.2 µg
  • UDP-GlcA: 0.625 mM
  • alpha -Lactose: 30 mM

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