Recombinant Human ST3GAL4 His-tag Protein, CF

• Assay Buffer: 50 mM Tris, 5 mM MnCl₂, pH 7.0
• Sialyltransferase Activity Kit (Catalog # EA002)
• Recombinant Human ST3GAL4 (rhST3GAL4) (Catalog # 10496-GT)
• CMP-Sialic Acid (Sigma, Catalog # C8271), 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 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 nmoles per well.
3. Prepare a reaction mixture containing 0.2 mM CMP-Sialic Acid, 36 mM alpha -Lactose and 2 µg/mL Coupling Phosphatase 2 (supplied in kit) in Assay Buffer.
4. Dilute rhST3GAL4 to 8 µg/mL in Assay Buffer. *Note: Perform single dilution step if possible to achieve concentration and avoid any acute mechanical stresses such as vortexing.
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 rhST3GAL4 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 room temperature 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:

• rhST3GAL4: 0.2 µg
• Coupling Phosphatase 2: 0.05 µg
• CMP-Sialic Acid: 0.1 mM
• alpha -Lactose: 18 mM