• Purity

  >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to transfer fucose from GDP-fucose to     N-Acetyllactosamine The specific activity is >1,000 pmol/min/μg, as measured under the described conditions.    
  See Activity Assay Protocol on .

• Source

  Chinese Hamster Ovary cell line, CHO-derived Thr33-Asn359

• Accession #

• N-terminal Sequence    
  Analysis

  Thr33

• Predicted Molecular Mass

  39 kDa

• SDS-PAGE

  40-50 kDa, reducing conditions

Assay Procedure

Materials

• Glycosyltransferase Activity Kit (Catalog # )

• 10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl₂, pH 7.5

• MnCl₂ (supplied in kit): 100 mM

• Recombinant Human Fucosyltransferase 9/FUT9 (rhFUT9) (Catalog # 9347-GT)

• GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water

• N-acetyl-Lactosamine (V-Labs, Catalog # GN204), 50 mM stock in deionized water

• 96-well Clear Plate (Catalog # )

• 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 0.16 mM GDP-Fucose, 0.6 mM Lactosamine, and 4 μg/mL Coupling Phosphatase 1 in 1X Assay Buffer.

5. Dilute rhFUT9 to 1 μg/mL in 1X Assay Buffer.

6. Load 50 μL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.

7. Load 25 μL of 1 μg/mL rhFUT9 into empty wells of the same plate as the curve. Include a Control containing 25 μL of 1X Assay Buffer.

8. Add 25 μL of the reaction mixture to all wells, excluding the standard curve. 

9. Seal plate and incubate at 37 °C for 20 minutes.

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:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

• rhFUT9: 0.025 μg

• Coupling Phosphatase 1: 0.1 μg

• GDP-Fucose: 0.08 mM

• Lactosamine: 0.3 mM

Background: Fucosyltransferase 9/FUT9

N-glycans, O-glycans and glycolipids 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 the leukocyte homing and 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 (2). FUT1 and FUT2 are alpha 1-2 fucosyltransferases and are responsible for ABO blood-group antigen synthesis. FUT8 is an alpha 1-6 fucosyltransferase that adds a fucose to the chitobiose core of N-glycans (3). FUT3, FUT4, FUT5, FUT6, FUT7 and FUT9 are alpha 1-3 or alpha 1-4 fucosyltransferases and are responsible for Lewis antigen generation. In particular, FUT9 synthesizes the Lewis X oligosaccharide (CD15) in the organ buds progressing in mesenchyma during embryogenesis and in mature granulocytes (4, 5). The activity of this enzyme has been measured with a phosphatase-coupled method (6).

• References:

1. Jafar-Nejad, H. et al. (2010) Glycobiology 20:931.

2. Becker, D.J. et al. (2003) Glycobiology 13:41R.

3. Lee, S.H. et al. (2006) J. Biochem. 139:391.

4. Kaneko M. et al. (1999) FEBS Lett. 452:237.

5. Brito, C. et al. (2008) Biochimie. 90:1279.

6. Wu, Z.L. et al. (2011) Glycobilogy 21:727.

• Entrez Gene IDs:

  10690 (Human); 14348 (Mouse); 84597 (Rat)

• Alternate Names:

  Alpha-(1,3)-Fucosyltransferase 9; alpha-(1,3)-fucosyltransferase; EC 2.4.1; EC 2.4.1.-; EC 2.4.1.65; fucosyltransferase 9 (alpha (1,3) fucosyltransferase); Fucosyltransferase 9; Fucosyltransferase IX; FucT-IX; Fuc-TIXFucT-IX; FUT9; Galactoside 3-L-fucosyltransferase