Recombinant Human ST3GAL6 Fc Chimera Protein, CF

• Assay Buffer: 25 mM Tris, 150 mM NaCl, 5 mM CaCl₂, 10 mM MnCl₂, pH 7.5
• Sialyltransferase Activity Kit (Catalog # EA002)
• Recombinant Human ST3GAL6 (rhST3GAL6) (Catalog # 10591-GT)
• CMP-Sialic Acid (Calbiochem, Catalog # 233264), 2.5 mM stock in deionized water
• N-Acetyllactosamine (Dextra Laboratories, Catalog # GN204), 50 mM 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 Sialyltransferase 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. 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 a reaction mixture containing 0.4 mM CMP-Sialic Acid, 8 mM N-Acetyllactosamine and 4 µg/mL Coupling Phosphatase 2 (supplied in kit) in Assay Buffer.
4. Dilute rhST3GAL6 to 20 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 20 µg/mL rhST3GAL6 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 the wells, excluding the standard curve.
8. Seal plate and incubate at 37 °C for 30 minutes.
9. Add 30 µL of the Malachite Green Reagent A (supplied in kit) 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 (supplied in kit) 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:

• rhST3GAL6: 0.5 µg
• Coupling Phosphatase 2: 0.1 µg
• CMP-Sialic Acid: 0.2 mM
• N-Acetyllactosamine: 4 mM