Recombinant Human Protein O-fucosyltransferase 1/POFUT1, CF SummaryLearn more about Fluorescent Glycan Labeling and Detection
Gly27-Leu384, with N-terminal HA (YPYDVPDYA) tag
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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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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 Buffer: 0.1 M Tris, 10 mM MnCl2, pH 7.0
- Coupling Enzyme Buffer: 0.1 M Tris, 5 mM CaCl2, pH 7.5
- Recombinant Human Protein O‑fucosyltransferase 1/POFUT1 (rhPOFUT1) (Catalog # 7409-GT)
- Donor Substrate: GDP-Fucose (Sigma, Catalog # G4401), 1.6 mM 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 Donor Substrate to 0.16 mM in Assay Buffer.
- Dilute rhPOFUT1 to 80 ng/µL in Assay Buffer.
- Combine equal volumes of 0.16 mM Donor Substrate and 80 ng/µL POFUT-1. Include a substrate blank containing Assay Buffer in place of 80 ng/µL POFUT-1.
- Incubate at 37 °C for 24 hours.
- Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
- Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Dilute Coupling Enzyme to 2 μg/mL in Coupling Enzyme Buffer.
- Load 50 µL of the incubated reactions and blanks into the plate in triplicates.
- Add 50 µL of 2 µg/mL Coupling Enzyme to the reaction wells and blanks, excluding the standard curve. Add 50 µL of the Coupling Enzyme Buffer to wells containing the phosphate standard curve. Mix and incubate for 10 minutes at room temperature.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 50 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.
- rhPOFUT1: 2 µg
- Coupling Enzyme: 0.1 µg
- Donor Substrate: 4 nmol
Background: Protein O-Fucosyltransferase 1/POFUT1
Fucose is typically found as a terminal modification of branched chain glycoconjugates, but also exists in direct O-linkage to serine or threonine residues of a number of cell surface and secreted proteins (1, 2). O-fucose was originally identified from human urine (3) and subsequently from an EGF repeat of urinary-type plasminogen activator (uPA) (4). The consensus sequence for O-fucose modification was determined to be C2XXGGS/TC3, where C2 and C3 are the second and third conserved cysteine residues of the EGF repeat, X is any amino acid, and S/T is the modified serine or threonine (5). O-Fucose exists on glycoproteins as either a monosaccharide (Fuc-O-Ser/Thr), a tetrasaccharide (NeuAc alpha 2, 3/6Gal beta 1, 4GlcNAc beta 1, 3Fuc-O-Ser/Thr), or a di- or trisaccharide intermediate in tetrasaccharide biosynthesis (5, 6). O-fucose glycans play important roles in Notch signaling (7). There are two protein O-fucosyltransferases, POFUT1 and POFUT2, and both enzymes are endoplasmic reticulum resident proteins with a short N-terminal cytoplasmic domain and a single pass transmembrane domain (type II membrane protein). For soluble expression of the protein, both the N-terminal transmembrane domain and the C-terminal ER retention sequence have been removed. POFUT1 is activated by manganese and has some hydrolase activity on the donor substrate GDP-Fucose. The hydrolase activity of the recombinant human POFUT1 was assayed using a malachite-based method (8).
- Martinez-Duncker, I. et al. (2003) Glycobiology 13:1c.
- Wang, Y. et al. (2001) ) J. Biol. Chem. 276:40338.
- Hallgren, P. et al. (1975) J. Biol. Chem. 250:5312.
- Kentzer, E.J. et al. (1990) Biochem. Biophys.Res. Commun. 171:401.
- Harris, R.J. and Spellman, M.W. (1993) Glycobiology 3:219.
- Moloney, D.J. et al. (2000) Nature 406:369.
- Stanley, P. and Guidos, C.J. (2009) Immunol. Rev. 230:201.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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Glycan Detection Reagents
Glycosyltransferase Activity Assays
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