Recombinant Human B4GalT7 His-tag Protein, CF

• 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

1. Prepare 1X Assay Buffer containing 10 mM MnCl2 by combining 10X stocks and diluting 10-fold with deionized water.
2. 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.
3. 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.
4. Prepare Reaction Mixture containing 0.5 mM UDP-Galactose and 20 mM D-Xylose in 1X Assay Buffer.
5. Dilute Coupling Phosphatase I (supplied in kit) to 10 µg/mL in 1X Assay Buffer.
6. Dilute rhB4GALT7 to 10 µg/mL in 1X Assay Buffer.
7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
8. 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.
9. Add 10 µL of 10 µg/mL Coupling Phosphatase I to all wells, excluding standard curve.
10. Start the reactions by adding 25 µL of Reaction Mixture to all wells, excluding the standard curve and curve blank. 
11. Seal plate and incubate at 37 °C for 30 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells used, including standard curve. Mix briefly.
13. Add 100 µL of deionized water to all wells used, including standard curve. Mix briefly.
14. Add 30 µL of the Malachite Green Reagent B to all wells used, including standard curve. Mix briefly.
15. Seal plate and incubate at room temperature for 20 minutes.
16. Read plate at 620 nm (absorbance) in endpoint mode.
17. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

• rhB4GALT7: 0.15 µg
• Coupling Phosphatase I: 0.1 µg
• UDP-Galactose: 0.25 mM
• D-Xylose: 10 mM

Scientific Data

Enzyme Activity View Larger

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.

SDS-PAGE View Larger

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.