Recombinant Human ST6GALNAC4 Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• Assay Buffer: 50 mM Tris, 10 mM MnCl₂ (supplied in kit), pH 7.0
• Recombinant Human ST6GALNAC4 (rhST6GALNAC4) (Catalog # 6876-GT)
• Donor Substrate: CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
• Acceptor Substrate: Fetuin (Sigma, Catalog # F3385), 50 mg/mL stock in diH20
• Sialyltransferase Activity Kit (Catalog # EA002)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute CMP-Sialic Acid to 2.5 mM in Assay Buffer.
2. Dilute Coupling Phosphatase 2 to 10 µg/mL in Assay Buffer.
3. Prepare reaction mixture by combining 75 µL of 2.5 mM CMP-Sialic Acid, 150 µL of 10 µg/mL Coupling Phosphatase 2, 75 µL of 50 mg/mL Fetuin, and 75 µL of Assay Buffer.
4. Dilute rhST6GALNAC4 to 0.8 µg/mL in Assay Buffer.
5. Dilute 1 mM Phosphate Standard by adding 40 µL to 360 µL of Assay Buffer for a 100 μM stock. This is the first point of the standard curve.
6. Continue 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 5.0 nmol per well.
7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
8. Load 25 µL of the 0.8 µg/mL rhST6GALNAC4 into the plate. Include a control containing 25 µL of Assay Buffer.
9. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
10. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
11. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix and incubate for 10 minutes at room temperature.
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 the control.

• rhST6GALNAC4: 0.02 µg
• Coupling Phosphatase 2: 0.1 µg
• Fetuin: 250 µg
• CMP-Sialic Acid: 0.25 mM