Recombinant Human Fucosyltransferase 2/FUT2 Protein, CF
Recombinant Human Fucosyltransferase 2/FUT2 Protein, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Ile35-His343, with an C-terminal 6-His tag
Analysis
Product Datasheets
7770-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.
7770-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, DTT and Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. 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, 10 mM CaCl2, pH 7.5
- Recombinant Human FUT2 (rhFUT2) (Catalog # 7770-GT)
- GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
- alpha -lactose (Sigma, Catalog # L2643), 0.3 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 1 mM Phosphate Standard provided by the Glycosyltransferase Kit to 100 µM by adding 40 µL of standard to 360 µL of Assay Buffer. This is the first point of the standard curve.
- Perform six additional one-half serial dilutions in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containg 100 mM alpha -lactose, 200 µM GDP-Fucose, and 4 µg/mL Coupling Phosphatase I in Assay Buffer.
- Dilute rhFUT2 to 1.0 µg/mL in Assay Buffer.
- 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 1 µg/mL rhFUT2 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture (step 3) to the wells, excluding the standard curve and curve blank.
- Cover the plate with a plate sealer and incubate at 37 °C for 20 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 inubate 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 Reaction:- rhFUT-2: 0.025 µg
- Coupling Phosphatase I: 0.1 µg
- alpha -Lactose: 50 mM
- GDP-Fucose: 100 µM
Background: Fucosyltransferase 2/FUT2
Glycans are frequently fucosylated at terminal sites. Therefore, fucose is often part of a sugar epitope with important biological function. Well‑known fucose‑containing glycans include Lewis and ABO blood group antigens. Lewis epitopes are key elements involved in leukocyte homing and the extravasation process, and thus are important for lymphocyte maturation and natural defense functions. Fucose‑containing glycans also play critical roles in cell signaling and development (1). More than 10 fucosyltransferases have been cloned, and most are Golgi‑resident type II membrane proteins (2). FUT3, FUT4, FUT5, FUT6, FUT7, and FUT9 are alpha 1‑3 or alpha 1‑4 fucosyltransferases and are responsible for Lewis antigen generation (3, 4, 5). FUT8 is the only alpha 1‑6 fucosyltransferase that adds a fucose to the chitobiose core of N‑glycans (6). FUT1 and FUT2 are galactoside alpha 1‑2 fucosyltransferases that generate H‑antigen, the precursor for ABO blood‑group antigen synthesis. In particular, FUT2 involves in generating soluble ABO antigen in saliva; therefore it is also called secretor blood group alpha 1‑2 fucosyltransferase (7). Absence of functional FUT2 is related to Crohn’s disease (8). The activity of this enzyme has been measured with a phosphatase‑coupled method (9).
- Jafar-Nejad, H. et al. (2010) Glycobiology 20:931.
- Becker, D.J. et al. (2003) Glycobiology 13:41R.
- Blander, J. M. et al. (1999) J. Immunol. 163:3746.
- Natsuka, S. et al. (1994) J. Biol. Chem. 269:16789.
- Sasaki, K. et al. (1994) J. Biol. Chem. 269:14730.
- Lee, S.H. et al. (2006) J. Biochem. 139:391.
- Kelly, R.J. et al. (1995) J. Biol. Chem. 270:4640.
- McGovern, D.P. et al. (2010) Hum. Mol. Genet. 19:3468.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.Citations for Recombinant Human Fucosyltransferase 2/FUT2 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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Rewiring the pneumococcal capsule pathway for investigating glycosyltransferase specificity and genetic glycoengineering
Authors: Su, T;Chua, WZ;Liu, Y;Fan, J;Tan, SY;Yang, DW;Sham, LT;
Science advances
Species: Bacteria
Sample Types: Whole Cells
Applications: Enzyme Activity -
Towards Mapping of the Human Brain N-Glycome with Standardized Graphitic Carbon Chromatography
Authors: J Helm, L Hirtler, F Altmann
Biomolecules, 2022-01-06;12(1):.
Species: Human
Sample Types: Small Molecule
Applications: Bioassay
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