Recombinant Porcine alpha3GalT/GGTA1 His-tag Protein, CF

• Glycosyltransferase Activity Kit (Catalog # EA001)
• Phosphate Buffer 1: 10X solution of 250 mM Tris, 100 mM CaCl₂, pH 7.5 (provided in kit)
• MnCl₂: 100 mM MnCl₂ in deionized water (provided in kit)
• Recombinant Porcine alpha 3GalT/GGTA1 His-tag (rpGGTA-1) (Catalog # 11261-GT)
  
• UDP-Galactose, 10 mM stock in deionized water
• alpha -Lactose, 0.3 M stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader

1. Prepare 1X Assay Buffer (25 mM Tris, 10 mM CaCl₂, 10 mM MnCl₂, pH 7.5) containing 10 mM MnCl₂ by combining equal volumes of Phosphate Buffer 1 and 100 mM MnCl₂ and diluting 5-fold with deionized water.
2. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
3. Complete the standard curve by performing six 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. Dilute Coupling Phosphatase 1 (provided in kit) to 10 µg/mL in 1X Assay Buffer.
5. Prepare a Reaction Mixture containing 0.8 mM UDP-Galactose, 40 mM alpha -Lactose, and 4 µg/mL Coupling Phosphatase 1 in 1X Assay Buffer.
6. Dilute rpGGTA-1 to 5 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 25 µL of 5 µg/mL rpGGTA-1 into empty wells of the same plate as the curve. Include a Control containing 25 µL of 1X Assay Buffer.
9. Start the reaction by adding 25 µL of Reaction Mixture to all wells, excluding standard curve.
10. Seal and incubate plate at 37 °C for 20 minutes.
11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
12. Add 100 µL of deionized water to all wells. Mix briefly.
13. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
14. Read plate at 620 nm (absorbance) in endpoint mode.
15. Calculate specific activity:
    
    

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

Per Reaction:

• rpGGTA-1 0.125 µg
• Coupling Phosphatase 1:  0.1 µg
• UDP-Galactose 0.4 mM
• alpha -Lactose: 20 mM