Recombinant Human

Fucosyltransferase 8/FUT8 Catalog Number: 5768-GT

DESCRIPTION Source Chinese Hamster Ovary cell line, CHO­derived human Fucosyltransferase 8/FUT8 protein Asp32­Lys575, with an N­terminal 6­His tag Accession # Q9BYC5

N­terminal Sequence His Analysis Predicted Molecular 64 kDa Mass

SPECIFICATIONS SDS­PAGE 58­62 kDa, reducing conditions

Activity Measured by its ability to hydrolyze the donor substrate GDP­fucose. The specific activity is >0.75 pmol/min/μg, as measured under the described conditions.

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

Purity >90%, by SDS­PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane. Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. See Certificate of Analysis for details.

Activity Assay Protocol Materials l Assay Buffer: 0.1 M MES, pH 7.0 l Recombinant Human Fucosyltransferase 8/FUT8 (rhFUT8) (Catalog # 5768­GT) l Coupling Enzyme: Recombinant Human CD39L3/ENTPD3 (rhCD39L3) (Catalog # 4400­EN) l Substrate: GDP­Fucose (Sigma, Catalog # G4401), 1.6 mM stock in deionized water l Malachite Green Phosphate Detection Kit (Catalog # DY996) l 96­well Clear Plate (Costar, Catalog # 92592) l Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

Assay 1. Dilute GDP­Fucose to 256 µM in Assay Buffer. 2. Dilute rhCD39L3 to 8 µg/mL in Assay Buffer. 3. Dilute rhFUT8 to 80 µg/mL in Assay Buffer. 4. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. 5. Prepare standard curve by performing seven one­half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well. 6. Prepare a reaction mixture by combining equal volumes of 8 µg/mL rhCD39L3 and 256 µM GDP­Fucose. 7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer. 8. Load 25 µL of the 80 µg/mL rhFUT8 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer. 9. Start the reaction by adding 25 µL of reaction mixture (step 6) to the wells, excluding the standard curve and curve blank. 10. Cover the plate with parafilm or a plate sealer and incubate at room temperature for 2 hours. 11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature. 12. Add 100 µL of deionized water to all wells. Mix briefly. 13. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature. 14. Read plate at 620 nm (absorbance) in endpoint mode. 15. Calculate specific activity: Phosphate released* (nmol) x (1000 pmol/nmol) Specific Activity (pmol/min/µg) = Incubation time (min) x amount of enzyme (µg) *Derived from the phosphate standard curve using linear or 4­parameter fitting and adjusted for Substrate Blank.

Final Assay Per Well: Conditions l rhFUT8: 2 µg l rhCD39L3: 0.1 µg l GDP­Fucose: 64 μM

PREPARATION AND STORAGE 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 freeze­thaw cycles. l 6 months from date of receipt, ­70 °C as supplied. l 3 months, ­70 °C under sterile conditions after opening.

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Recombinant Human

Fucosyltransferase 8/FUT8 Catalog Number: 5768-GT

BACKGROUND N­glycans, O­glycans and glycolipids are frequently fucosylated at terminal sites. Therefore, fucose is often part of a sugar epitope with an important biological function. Well­known fucose­containing glycans include Lewis and ABO blood group antigens. Lewis epitopes are key elements involved in leukocyte homing and extravasation and thus are important for lymphocyte maturation and natural defense functions. Fucose­containing glycans also play critical roles in cell signaling and development (1). More than 10 fucosyltransferases have been cloned and all of them are Golgi­resident type II membrane proteins (2). FUT1 and FUT2 are α1­ 2 fucosyltransferases and are responsible for ABO blood­group antigen synthesis. FUT3, FUT4, FUT5, FUT6, FUT7 and FUT9 are α1­3 or α1­4 fucosyltransferases and are responsible for Lewis antigen generation (3, 4, 5). FUT8 is the only α1­6 fucosyltransferase that adds a fucose to the chitobiose core of N­glycans (6). The α1,6­fucosylation of N­glycan in human IgG1 was reported to suppress antibody­dependent cellular cytotoxicity (7, 8). Disruption of the FUT8 gene induced severe growth retardation, emphysema, and death during postnatal development in mice. The absence of α1,6­fucosylation on transforming growth factor­β1 (TGF­β1) receptors was found to be involved in the mouse phenotypes (9). The activity of this enzyme has been measured using a phosphatase­coupled assay (10).

References: 1. Jafar­Nejad, H. et al. (2010) Glycobiology 20:931. 2. Becker, D.J. et al. (2003) Glycobiology 13:41R. 3. Blander, J. M. et al. (1999) J. Immunol. 163:3746. 4. Natsuka, S. et al. (1994) J. Biol. Chem. 269:16789. 5. Sasaki, K. et al. (1994) J. Biol. Chem. 269:14730. 6. Lee, S.H. et al. (2006) J. Biochem. 139:391. 7. Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733. 8. Shinkawa, T. et al. (2003) J. Biol. Chem. 278:3466. 9. Wang, X. et al. (2005) Proc. Natl. Acad. Sci. U. S. A. 102:15791. 10. Wu, Z.L., et al. (2010) Glycobiology doi: 10.1093/glycob/cwq187.

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