Recombinant Human B4GalT7 His-tag Protein, CF Summary
The specific activity is >120 pmol/min/μg, as measured under the described conditions.
Ser52-Ser327, with C-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)
- 10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl2, pH 7.5
- MnCl2 (supplied in kit): 100 mM
- Recombinant Human Beta-1,4-Galactosyltransferase 7/B4GalT7 His-tag (rhB4GALT7) (Catalog # 10663-GT)
- D-Xylose (V-labs, Catalog # BX53), 100 mM stock in deionized water
- UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare 1X Assay Buffer containing 10 mM MnCl2 by combining 10X stocks and diluting 10-fold with deionized water.
- Prepare a standard curve from the 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock.
- Perform six additional one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare Reaction Mixture containing 0.5 mM UDP-Galactose and 20 mM D-Xylose in 1X Assay Buffer.
- Dilute Coupling Phosphatase I (supplied in kit) to 10 µg/mL in 1X Assay Buffer.
- Dilute rhB4GALT7 to 10 µg/mL in 1X Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Load 15 µL of 10 µg/mL rhB4GALT7 into empty wells of the same plate as the curve. Include a Control containing 15 μL of 1X Assay Buffer.
- Add 10 µL of 10 µg/mL Coupling Phosphatase I to all wells, excluding standard curve.
- Start the reactions by adding 25 µL of Reaction Mixture to all wells, excluding the standard curve and curve blank.
- Seal plate and incubate at 37 °C for 30 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells used, including standard curve. Mix briefly.
- Add 100 µL of deionized water to all wells used, including standard curve. Mix briefly.
- Add 30 µL of the Malachite Green Reagent B to all wells used, including standard curve. Mix briefly.
- Seal plate and incubate at room temperature for 20 minutes.
- 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:
- rhB4GALT7: 0.15 µg
- Coupling Phosphatase I: 0.1 µg
- UDP-Galactose: 0.25 mM
- D-Xylose: 10 mM
B4GalT7 attaches the first galactose in the common carbohydrate-protein (GlcA-beta -1,3-Gal-beta -1,3-Gal-beta -1,4-Xyl-beta1-O-Ser) linkage found in proteoglycans.
1 μg/lane of Recombinant Human B4GalT7 His-tag (Catalog # 10663-GT) was resolved with SDS-PAGE under reducing (R) conditions and visualized by silver staining, showing bands at 37-41 kDa.
Background: Beta-1,4-Galactosyltransferase 7/B4GalT7
There are seven beta-1,4-galactosyltransferases that transfer galactose in a beta-1,4 linkage to acceptor sugars including GlcNAc, and Glc, and Xyl. B4GalT7, also known as galactosyltransferase I, catalyzes the addition of the first galactose to the glycosaminoglycan-protein tetrasaccharide linkage (GlcA-beta -1,3-Gal-beta -1,3-Gal-beta -1,4-Xyl-beta1-O-Ser) in proteoglycans (1). Proteoglycans are structural components of the extracellular matrix that is found between cells in connective tissues (2). B4GalT7 resides in cis-Golgi apparatus and is a type II membrane protein and differs from the other six B4GalTs for lacking a conserved Cys residue (3). Mutations in the gene of B4GalT7 have been associated with Ehlers-Danlos syndrome (4, 5) and Larsen syndrome (6), likely due to reduced glycosaminoglycan synthesis in decorin and biglycan, which further results in delayed wound repair, altered migration, adhesion and contractility of patient fibroblasts (7, 8). The activity of this enzyme has been measured with a phosphatase-coupled method (9).
- Almeida, R. et al. (1999) J. Biol. Chem. 274: 26165.
- Iozzo, RV and Schaefer, L (2015) Matrix Biology 42:11.
- Tsutsui, Y., Ramakrishnan, B. and Qasba, P.K. (2013) J. Biol. Chem. 288:31963.
- Furukawa, K, and Okajima, T (2002) Biochim. Biophys. Acta 1573:377.
- Okajima, T. et al. (1999) J. Biol. Chem. 274:28841.
- Cartault, F. et al. (2015) Eur. J. Hum. Genet. 23:49.
- Seidler, D.G. et al. (2006). J. Mol. Med. 84:583.
- Götte, M. et al. (2008). Hum. Mol. Genet. 17:996.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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