Recombinant Human B4GalT1 Protein, CF
Recombinant Human B4GalT1 Protein, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Gly44-Ser398, with an N-terminal 6-His tag
Analysis
Product Datasheets
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.
3609-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 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
- Dilute UDP-Galactose to 0.5 mM in Assay Buffer.
- Dilute Coupling Phosphatase 1 to 20 µg/mL in Assay Buffer.
- Dilute GlcNAc to 50 mM in Assay Buffer.
- Prepare Reaction Mixture by combining equal volumes of 0.5 mM UDP-GalNAc, 20 µg/mL Coupling Phosphatase 1, and 50 mM GlcNAc.
- Dilute rhB4GalT1 to 0.75 µg/mL in Assay Buffer.
- 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.
- 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 20 µL of the 0.75 µg/mL rhB4GalT1 into the plate. Include a Control containing 20 µL of Assay Buffer.
- Start the reaction by adding 30 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
- Seal plate and incubate at room temperature for 15 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- 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 Control.
Per Well:- rhB4GalT1: 0.015 µg
- Coupling Phosphatase 1: 0.2 µg
- UDP-Galactose: 5 nmol
- GlcNAc: 500 nmol
Reconstitution Calculator
Background: beta-1,4-Galactosyltransferase 1/B4GalT1
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).
- Amado, M. et al. (1999) Biochim. Biophys. Acta. 1473:35.
- Yamaguchi, N and Fukuda, M.N. (1995) J. Biol. Chem. 270:12170.
- Appert, H.E. et al. (1986) Biochem. Biophys. Res. Commun. 138:224.
- Lopez, L.C. et al. (1991) J. Biol. Chem. 266:15984.
- Mengle-Gaw, L. et al. (1991) Biochem. Biophys. Res. Commun. 176:1269.
- Hansske, B. et al. (2002) J. Clin. Invest. 109:725.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citation for Recombinant Human B4GalT1 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.
1 Citation: Showing 1 - 1
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Mice overexpressing beta-1,4-Galactosyltransferase I are resistant to TNF-induced inflammation and DSS-induced colitis.
Authors: Vanhooren V, Vandenbroucke R, Dewaele S, Van Hamme E, Haigh J, Hochepied T, Libert C
PLoS ONE, 2013-12-05;8(12):e79883.
Species: Mouse
Sample Types: Whole Tissue
Applications: Bioassay
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