Recombinant Human Fucosyltransferase 11/FUT11 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Fucosyltransferase 11/FUT11 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the donor substrate GDP-fucose. The specific activity is >4 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived human Fucosyltransferase 11/FUT11 protein
  Gly25-Leu492, with an N-terminal 6-His tag
• Accession #: AAH36037
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: FUT11
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 54 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: 65-73 kDa, reducing conditions

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 silver stain
• Assay Procedure:
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl₂, pH 7.5
  • MnCl₂ (supplied in kit): 100 mM
  • Recombinant Human Fucosyltransferase 11/FUT11 (rhFUT11) (Catalog # 5964-GT)
  • GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer containing 10 mM MnCl₂ by combining 10X stocks and diluting 10-fold with deionized water.
  2. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Prepare reaction mixture containing 0.2 mM GDP-Fucose and 8 µg/mL Coupling Phosphatase 1 in Assay Buffer.
  5. Dilute rhFUT11 to 80 ng/µL in Assay Buffer.
  6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  7. Load 25 µL of 80 ng/µL rhFUT11 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  8. Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
  9. Seal plate and incubate at room temperature for 1 hour.
  10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate sealed plate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the phosphate standard curve using linear fitting and adjusted for Control.

  Per Reaction:
  • rhFUT11: 2 µg
  • Coupling Phosphatase 1: 0.2 µg
  • GDP-Fucose: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Fucosyltransferase 11/FUT11 Protein, CF

• alpha-(1,3)-fucosyltransferase 11
• EC 2.4.1
• EC 2.4.1.-
• fucosyltransferase 11 (alpha (1,3) fucosyltransferase)
• Fucosyltransferase 11
• Fucosyltransferase XI
• Fuc-TXI
• fucT-XI
• FUT11
• Galactoside 3-L-fucosyltransferase 11
• MGC119338
• MGC119339
• MGC33202

Background

Fucose is frequently found at terminal sites on various glycans and is essential for the generation of many sugar epitopes. Well-known fucose containing glycans include Lewis and ABO blood group antigens. Lewis epitopes are key elements involved in leukocyte homing and extravasation process, thus are essential for lymphocyte maturation and natural defense functions (1). O-fucosylation on Notch receptor is found to be essential for its signaling function (2). So far, 11 carbohydrate specific fucosyltransferases are found in humans (3). FUT1 and FUT2 are alpha 1-2 fucosyltransferases and are responsible for ABO blood group antigen synthesis (4). FUT3, FUT4, FUT5, FUT6, FUT7 and FUT9 are alpha 1-3/4 fucosyltransferases and are responsible for Lewis antigen generation (5). FUT10 and FUT11 are newly cloned alpha 1-3 fucosyltransferases that are distinct from the alpha 1-3/4 fucosyltransferase subfamily and are able to introduce fucose to the innermost core GlcNAc of the N-glycan on conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates (6). Predicted as a type II transmembrane protein and a Golgi resident enzyme, the exact function of this enzyme needs to be further characterized. The activity of this enzyme has been measured with a phosphatasecoupled method (7).

1. Weston, B. W. et al. (1992) J. Biol.Chem. 267:4152.
2. Stahl, M. et al. (2008) J. Biol.Chem. 283:13638.
3. Becker, D.J. et al. (2003) Glycobiology 13:41R.
4. Kelly, R. J. et al. (1995) J. Biol.Chem. 270:4640.
5. Weston, B. W. et al. (1992) J. Biol.Chem. 267:24575.
6. Mollicone, R. et al. (2009) J. Biol. Chem. 284:4723.
7. Wu, Z.L. et al. (2011) Glycobilogy 21:727.

Publications for Fucosyltransferase 11/FUT11 (5964-GT)(1)

Publication using 5964-GT

• Wu Z, Zhou H, Ethen C, N Reinhold V Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase. Biochem Biophys Res Commun, 2016-03-21;473(2):524-9. 2016-03-21 [PMID: 27012207] (Bioassay)

Bioinformatics

• Gene Symbol: FUT11
• Uniprot:
  • AAH36037()