Recombinant Human B3GNT6 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

Order Details

Recombinant Human B3GNT6 Protein, CF Summary

Details of Functionality
Measured by its ability to transfer GlcNAc from UDP-GlcNAc to 4-nitrophenyl-alpha -D-galactosaminide. The specific activity is >35 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human Beta-1,3-N-Acetylglucosaminyltransferase 6/B3GNT6 protein
Gln32-Ser384, with an N-terminal human CD33 signal sequence and 6-His tag
Accession #
N-terminal Sequence
His
Protein/Peptide Type
Recombinant Enzymes
Gene
B3GNT6
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

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
43-55 kDa, reducing conditions
Publications
Read Publication using
6505-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 Colloidal Coomassie® Blue stain at 5 μg per lane.
Assay Procedure
  • Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl2, 5 mM CaCl2, 20% DMSO, pH 7.5
  • Recombinant Human beta -1,3-N-acetylglucosaminyltransferase 6/B3GNT6 (rhB3GNT6) (Catalog # 6505-GT)
  • Coupling Enzyme: Recombinant Human CD39L3/ENTPD3 (rhCD39L3) (Catalog # 4400-EN)
  • UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% EtOH (v/v)
  • 4-Nitrophenyl N-acetyl-alpha -D-galactosaminide (4-NP-GalNAc) (Sigma, Catalog # N4264), 15 mM stock in DMSO
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute UDP-GlcNAc to 1.2 mM in Assay Buffer.
  2. Dilute 4-NP-GalNAc to 3.6 mM in Assay Buffer.
  3. Dilute rhCD39L3 to 6 μg/mL in Assay Buffer.
  4. Prepare reaction mixture by combining equal volumes of 1.2 mM UDP-GlcNAc, 3.6 mM 4-NP-GalNAc, and 6 μg/mL rhCD39L3.
  5. Dilute rhB3GNT6 to 12 µg/mL in Assay Buffer.
  6. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 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 25 µL of the 12 µg/mL rhB3GNT6 into the plate. Include a substrate blank containing 25 µL of Assay Buffer.
  10. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  11. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 60 minutes.
  12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  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 Substrate.

Per Reaction:
  • rhB3GNT6: 0.300 µg
  • rhCD39L3: 50 ng
  • UDP-GlcNAc: 0.2 mM
  • 4-NP-GalNAc: 0.6 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 B3GNT6 Protein, CF

  • B3GNT6
  • B3Gn-T6
  • beta-1,3-Gn-T6
  • Beta-1,3-N-acetylglucosaminyltransferase 6
  • beta-1,3-N-acetylglucosaminyltransferase protein
  • Beta3Gn-T6
  • BGnT-6
  • Core 3 synthase
  • EC 2.4.1.-
  • IMAGE:4907098
  • MGC119334
  • MGC119336
  • MGC119337
  • UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 6 (core 3 synthase)
  • UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 6

Background

beta -1,3-N-acetylglucosaminyltransferase 6 (B3GNT6) is also known as core 3 synthase due to its role in synthesis of the core 3 structure (GlcNAc beta 1‑3Gal‑NAc alpha 1‑serine/threonine), an important precursor in the biosynthesis of mucin-type glycoproteins in digestive organs (1). Its expression is restricted to the stomach, colon and small intestine, where the core 3 structure is present. Down-regulation of the enzyme was found in gastric and colorectal carcinomas and it was suggested that it may be useful as a marker to distinguish between benign adenomas and premalignant lesions (2, 3). Prostate cancer cells transfected with core 3 synthase exhibited reduced migration and invasion compared with mock-transfected cells (4). When inoculated into nude mice, the transfected cells produced smaller tumors without metastasis in contrast to the robust tumor formation and metastasis observed in mock-transfected cells (4). Like other members of the beta ‑1,3‑N‑acetylglucosaminyltransferase family, B3GNT6 is a Golgi-resident single-pass type II membrane protein.The activity of this enzyme has been measured with a phosphatase-coupled method (5).
  1. Iwai, T. et al. (2002) J. Biol. Chem. 277:12802.
  2. Iwai, T. et al. (2005) Proc. Natl. Acad. Sci. USA 102:4572.
  3. Vavasseur, F. et al. (1995) Glycobiology 5:351.
  4. Lee, S.H. et al. (2009) J. Biol. Chem. 284:17157.
  5. Wu, Z.L. et al. (2010) Glycobiology doi: 10.1093/glycob/cwq187.

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Publications for Beta-1,3-N-Acetylglucosaminyltransferase 6/B3GNT6 (6505-GT)(1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: Click Chemistry.


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Bioinformatics

Gene Symbol B3GNT6
Uniprot