Recombinant Human Fucosyltransferase6/FUT6 Protein, CF
Recombinant Human Fucosyltransferase6/FUT6 Protein, CF Summary
Product Specifications
Arg35-Thr359
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.
8959-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| 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
- 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 6/FUT6 (rhFUT6) (Catalog # 8959-GT)
- GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water
- Lactosamine (V-Labs, Catalog # GN204), 50 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer containing 10 mM MnCl2 by combining 10X stocks and diluting 10 fold with deionized water.
- 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.
- 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.
- Prepare reaction mixture containing 0.16 mM GDP-Fucose, 0.6 mM Lactosamine, and 4 µg/mL Coupling Phosphatase 1 in 1X Assay Buffer.
- Dilute rhFUT6 to 4 ng/µL in 1X Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Load 25 µL of 4 ng/µL rhFUT6 into empty wells of the same plate as the curve. Include a Control containing 25 μL of 1X Assay Buffer.
- Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
- Seal plate 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 incubate sealed plate 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.
- rhFUT6: 0.1 µg
- Coupling Phosphatase 1: 0.1 µg
- GDP-Fucose: 0.08 mM
- Lactosamine: 0.3 mM
Reconstitution Calculator
Background: Fucosyltransferase 6/FUT6
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. Among these enzymes, FUT6 probably has the most diverse substrate specificity. It is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. This enzyme shares 85% amino acid sequence identity with FUT3 and 89% identity with FUT5 but differs substantially in its acceptor substrate specificity. The activity of this enzyme is measured with a phosphatase-coupled method (5).
- Jafar-Nejad, H. et al. (2010) Glycobiology 20:931.
- Becker, D.J. et al. (2003) Glycobiology 13:41R.
- Lee, S.H. et al. (2006) J. Biochem. 139:391.
- Weston, B.W. et al. (1992) J. Biol. Chem. 267:24575.
- Wu, Z.L. et al. (2011) Glycobilogy 21:727.
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