Recombinant Human B3GNT2 Protein, CF
Recombinant Human B3GNT2 Protein, CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Lys29-Cys397, with N-terminal 6-His tag
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.
3960-GT
| 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 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 2/B3GNT2 (rhB3GNT2) (Catalog # 3960-GT)
- UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
- N-acetyllactosamine (V-Labs, Catalog # GN204), 50 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.
- Dilute UDP-GlcNAc to 1.8 mM in Assay Buffer.
- Dilute N-acetyllactosamine to 6 mM in Assay Buffer.
- Dilute Coupling Phosphatase I to 6 ng/µL in Assay Buffer.
- Prepare reaction mixture by combining equal volumes of 1.8 mM UDP-GlcNAc, 6 mM N-acetyllactosamine, and 6 ng/µL Coupling Phosphatase I.
- Dilute rhB3GNT2 to 8 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 the 8 ng/µL rhB3GNT2 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 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 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:- rhB3GNT2: 0.2 µg
- Coupling Phosphatase I: 0.05 µg
- N-acetyllactosamine: 1 mM
- UDP-GlcNAc: 0.3 mM
Reconstitution Calculator
Background: Beta-1,3-N-Acetylglucosaminyltransferase 2/B3GNT2
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 (1-5). 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‑acetylglucosaminyltransferases 2 or beta 3GNT2 prefers the substrate lacto-N-neotetraose and is involved in the biosynthesis of poly‑N‑acetyllactosamine chains (6). The enzymatic activity of the recombinant protein was determined using a phosphatase-coupled assay (7).
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Seko, A. and Yamashita, K. (2005) Glycobiology 15:943.
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Kataoka, K. and Huh, N.-H. (2002) Biochem. Biophys. Res. Commun. 294:843.
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Iwai,T. et al. (2002) J. Biol. Chem. 277:12802.
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Togayachi, A. et al. (2001) J. Biol. Chem. 276:22032.
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Sasaki, K. et al. (1997) Proc. Natl. Acad. Sci. USA 94:14294.
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Shiraishi, N. et al. (2001) J. Biol. Chem. 276:3498.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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