Recombinant Human B3GAT1 Protein, CF Summary
His25-Ile334, with N-terminal 6-His tag
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 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.
- Glycosyltransferase Activity Kit (Catalog # EA001)
- Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM MnCl2, 5 mM CaCl2, pH 7.0
- Recombinant Human beta -1,3-Glucuronyltransferase 1/B3GAT1 (rhB3GAT1) (Catalog # 8560-GT)
- UDP-GlcA (Sigma, Catalog # U5625), 10 mM stock in deionized water.
- alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- 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. 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 Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containing 1.25 mM UDP-GlcA, 60 mM alpha -Lactose, and 8 ng/μL Coupling Phosphatase 1 in Assay Buffer.
- Dilute rhB3GAT1 to 2 ng/µL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 25 µL of 2 ng/µL rhB3GAT1 into empty wells of the same plate as the curve. Include a Control containing 25 μL of 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.Per Reaction:
- rhB3GAT1: 0.050 µg
- Coupling Phosphatase 1: 0.2 µg
- UDP-GlcA: 0.625 mM
- alpha -Lactose: 30 mM
Background: beta-1,3-Glucuronyltransferase 1/B3GAT1
B3GAT1 is a key enzyme involved in human natural killer1 (HNK1) epitope synthesis. It adds a glucuronic residue to the terminal lactosamine residue (Gal beta 14GlcNAc) of a glycoprotein or glycolipid, which can be further sulfated to become the HNK1 epitope, a unique trisaccharide structure, HSO3-3GlcA beta 1-3Gal beta 1-4GlcNAc (1, 2). The enzyme activity was found to be enhanced in the presence of sphingomyelin and phosphatidylinositol (3). The HNK1 carbohydrate epitope is characteristically expressed on a series of cell adhesion molecules in addition to some glycolipids in the extracellular matrix and on the cell surface in the nervous system, where it is involved in cell-cell and cell-substratum interaction and recognition during the development of the nervous system (4). Like most known glycosyltransferases, B3GAT1 is a type II Golgi-resident transmembrane protein with a short N-terminal cytoplasmic domain and a single pass transmembrane domain followed by an enzymatic domain in the lumen of Golgi apparatus. The enzyme activity was assayed using a phosphatase-coupled method (5).
- Terayama, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94:6093.
- Shogo, O. et al. (1992) J. Biol. Chem. 267: 22711.
- Kakuda, S. et al. (2005) Glycobiology 2:203.
- Bollensen, E. and Schachner, M. (1987) Neurosci Lett. 82:77.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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