Recombinant Human B3GalT5 His-tag Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

•  Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MgCl₂, 10 mM MnCl₂, pH 7.5
•  Glycosyltransferase Activity Kit  (Catalog # EA001)
•  Recombinant Human B3GalT5 (rhB3GalT5) (Catalog # 10555-GT)
•  Donor Substrate: UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
•  Acceptor Substrate: GlcNAc (N-Acetyl-alpha -D-glucosamine) (Millipore, Catalog # 1079), 1 M stock in deionized water
•  96-well Clear Plate  (Catalog # DY990)
•  Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit 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.
2. 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 nmoles per well.
3. Prepare reaction mixture containing 0.4 mM UDP-Galactose, 200 mM GlcNAc and 4 µg/mL Coupling Phosphatase 1 (supplied in kit) in Assay Buffer.
4. Dilute rhB3GalT5 to 8 µg/mL in Assay Buffer.
5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
6. Load 25 µL of 8 µg/mL rhB3GalT5 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
7. Add 25 µL of reaction mixture to all wells, excluding the standard curve.
8. Seal plate and incubate at 37 °C for 20 minutes.
9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
10. Add 100 µL of deionized water to all wells. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate sealed plate for 20 minutes at room temperature.
12. Read plate at 620 nm (absorbance) in endpoint mode.
13. Calculate specific activity:

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

• rhB3GalT5: 0.2 µg
• Coupling Phosphatase 1: 0.1 µg
• UDP-Galactose: 0.2 mM
• GlcNAc: 100 mM