Recombinant Human B3GNT6 Protein, CF
Recombinant Human B3GNT6 Protein, CF Summary
Product Specifications
Gln32-Ser384, with an N-terminal 6-His tag
Analysis
Product Datasheets
6505-GT
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
6505-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.
|
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
- Dilute UDP-GlcNAc to 1.2 mM in Assay Buffer.
- Dilute 4-NP-GalNAc to 3.6 mM in Assay Buffer.
- Dilute rhCD39L3 to 6 μg/mL in Assay Buffer.
- Prepare reaction mixture by combining equal volumes of 1.2 mM UDP-GlcNAc, 3.6 mM 4-NP-GalNAc, and 6 μg/mL rhCD39L3.
- Dilute rhB3GNT6 to 12 µg/mL in Assay Buffer.
- 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.
- 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.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 25 µL of the 12 µg/mL rhB3GNT6 into the plate. Include a substrate blank containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 60 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 100 µL of deionized water to all wells. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- 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
Background: Beta-1,3-N-Acetylglucosaminyltransferase 6/B3GNT6
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).
- Iwai, T. et al. (2002) J. Biol. Chem. 277:12802.
- Iwai, T. et al. (2005) Proc. Natl. Acad. Sci. USA 102:4572.
- Vavasseur, F. et al. (1995) Glycobiology 5:351.
- Lee, S.H. et al. (2009) J. Biol. Chem. 284:17157.
- Wu, Z.L. et al. (2010) Glycobiology doi: 10.1093/glycob/cwq187.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.Citations for Recombinant Human B3GNT6 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila
Authors: B Trastoy, A Naegeli, I Anso, J Sjögren, ME Guerin
Nat Commun, 2020-09-24;11(1):4844.
Species: E. coli
Sample Types: Peptide
Applications: Bioassay -
Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.
Authors: Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R
Glycobiology, 2018-02-01;0(0):.
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Click Chemistry
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