Recombinant Mouse alpha3GalT/GGTA1 Protein, CF

• Assay Buffer: 50 mM MES, 20 mM MnCl₂, pH 7.0
• Recombinant Mouse alpha 3GalT/GGTA1 (rmGGTA1) (Catalog # 7887-GT)
• UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
• alpha -Lactose (Sigma, Catalog # L2643), 0.3 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

1. Dilute 1 mM Phosphate Standard provided by the Universal Sulfotransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
2. Prepare 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 nmol per well.
3. Prepare reaction mixture containing 0.8 mM UDP‑Galactose, 8 µg/mL Coupling phosphatase 1, and 40 mM alpha -Lactose in Assay Buffer.
4. Dilute rmGGTA1 to 1.2 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 the 1.2 µg/mL rmGGTA1 into the plate. Include a Control containing 25 µL of Assay Buffer.
7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
8. Cover the plate with a plate sealer 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.
11. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate 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.

Per Reaction:

• rmGGTA1: 0.03 µg
• Coupling phosphatase 1: 0.2 µg
• UDP-Galactose: 0.4 mM
• alpha -Lactose: 20 mM