Recombinant Human B3GAT1 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.

• Glycosyltransferase Activity Kit (Catalog # EA001)
• Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM MnCl₂, 5 mM CaCl₂, pH 7.0
• Recombinant Human beta -1,3-Glucuronyltransferase 1/B3GAT1 (rhB3GAT1) (Catalog # 8560-GT)
• UDP-GlcA (Sigma, Catalog # U5625), 10 mM stock in deionized water.
• alpha -Lactose (Sigma, Catalog # L2643), 0.3 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 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. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
3. Prepare reaction mixture containing 1.25 mM UDP-GlcA, 60 mM alpha -Lactose, and 8 ng/μL Coupling Phosphatase 1 in Assay Buffer.
4. Dilute rhB3GAT1 to 2 ng/µL 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 2 ng/µL rhB3GAT1 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
7. Add 25 µL of the 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.

• rhB3GAT1: 0.050 µg
• Coupling Phosphatase 1: 0.2 µg
• UDP-GlcA: 0.625 mM
• alpha -Lactose: 30 mM