Recombinant Human B4GalT2 Protein, CF
Recombinant Human B4GalT2 Protein, CF Summary
Product Specifications
Ser43-Gly372, with C-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.
7530-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 MnCl2, 10 mM CaCl2, 65 mM NaCl, pH 7.5
- Recombinant Human beta ‑1,4‑Galactosyltransferase 2/B4GalT2 (rh beta 4GalT2) (Catalog # 7530-GT)
- UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
- D-(+)-Glucose (Sigma, Catalog # G5767), 2 M 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 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.
- Continue 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 5.0 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 rh beta 4GalT2 to 16 µg/mL.
- Prepare a Reaction Mixture composed of 0.4 M Glucose, 1 mM UDP-Galactose, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
- Load 25 µL of the 16 µg/mL rh beta 4GalT2 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Start the reaction by adding 25 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
- Cover the plate with a plate sealer and incubate at room temperature 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 Well:- rh beta 4GalT2: 0.4 µg
- Coupling Phosphatase 1: 0.1 µg
- UDP-Galactose: 0.5 mM
- Glucose: 200 mM
Reconstitution Calculator
Background: beta-1,4-Galactosyltransferase 2/B4GalT2
There are seven beta -1,4-galactosyltransferases that transfer galactose in a beta -1,4 linkage to acceptor sugars including GlcNAc, Glc, and Xyl. By sequence similarity, the beta 4GalTs form four groups: beta 4GalT1 and beta 4GalT2, beta 4GalT3 and beta 4GalT4, beta 4GalT5 and beta 4GalT6, and beta 4GalT7 (1). All of these enzymes are expressed as type II membrane proteins in Golgi apparatus except beta 4GalT1, which can be expressed as a secreted form in lactating mammary tissues due to an alternative transcription initiation site (2, 3). beta 4GalT2 is responsible for the synthesis of complex-type N-linked oligosaccharides in many glycoproteins as well as the carbohydrate moieties of glycolipids (4). It can also produce lactose. Its substrate specificity is affected by alpha -lactalbumin, but it is not expressed in lactating mammary tissue (5). Recently, beta 4GalT2 has been shown to form a complex with GlcAT-P to synthesize HNK-1 carbohydrate in the nervous system (6). The activity of this enzyme has been measured with a phosphatase-coupled method (7).
- Amado, M. et al. (1999) Biochim. Biophys. Acta. 1473:35.
- Appert, H.E. et al. (1986) Biochem. Biophys. Res. Commun.138:224.
- Mengle-Gaw,L. et al. (1991) Biochem. Biophys. Res.Commun. 176:1269.
- Guo, S. et al. (2001) Glycobiology 11:813.
- Almeida, R. et al. (1997) J. Biol. Chem. 272:31979.
- Kouno, T. et al. (2011) J. Biol. Chem. 286:31337.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.FAQs
No product specific FAQs exist for this product, however you may
View all Proteins and Enzyme FAQsReviews for Recombinant Human B4GalT2 Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human B4GalT2 Protein, CF and earn rewards!
Have you used Recombinant Human B4GalT2 Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
