Recombinant Human B3GNT4 Protein, CF
Recombinant Human B3GNT4 Protein, CF Summary
Product Specifications
Ala56-Arg378, with a C-terminal 6-His tag
Analysis
Product Datasheets
7696-GT
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.
7696-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
- Assay Buffer: 25 mM Tris, 10 mM CaCl2, 10 mM MnCl2 (supplied in kit), pH 7.5
- Recombinant Human beta -1,3-N-Acetylglucosaminyltransferase 4/B3GNT4 (rhB3GNT4) (Catalog # 7696‑GT)
- UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
- beta -Lactose (Sigma, Catalog # L3750), 250 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 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.
- Prepare reaction mixture containing 80 mM beta -Lactose, 2 mM UDP-GlcNAc, and 4 µg/mL Coupling Phosphatase I in Assay Buffer.
- Dilute rhB3GNT4 to 12 µg/mL 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 the 12 µg/mL rhB3GNT4 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
- Seal the plate with parafilm or a plate sealer and incubate at 37 ºC for 2 hours.
- 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 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:- rhB3GNT4: 0.3 µg
- Coupling Phosphatase I: 0.1 µg
- beta -Lactose: 40 mM
- UDP-GlcNAc: 1.0 mM
Background: Beta-1,3-N-Acetylglucosaminyltransferase 4/B3GNT4
beta 1,3-Linked GlcNAc residues are present in the backbone of various biologically important glycans which are involved in many essential biological functions such as keratan sulfate synthesis in corneal tissue (1). The addition of such residues are catalyzed by a family of beta 1,3-N-acetylglucosaminyltransferases, that includes at least eight members (15). All of them are type II Golgi resident transmembrane proteins and have high homology to the beta 1,3-galactosyltransferase family. beta 1,3-N-acetylglucosaminyltransferase 4 or beta 3GNT4 is involved in polylactosamine synthesis and is mainly expressed in brain tissues such as whole brain, hippocampus, amygdala, cerebellum and caudate nucleus (6). The enzymatic activity of the recombinant protein was determined using a phosphatase coupled assay (7).
- Seko, A. and Yamashita, K. (2005) Glycobiology 15:943.
- Kataoka, K. and Huh, N.H. (2002) Biochem. Biophys. Res. Commun. 294:843.
- Iwai,T. et al. (2002) J. Biol. Chem. 277:12802.
- Togayachi, A. et al. (2001) J. Biol. Chem. 276:22032.
- Sasaki, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94:14294.
- Shiraishi, N. et al. (2001) J. Biol. Chem. 276:3498.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Product Specific Notices
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