Recombinant Human ST6GALNAC6 Protein, CF

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

• Assay Buffer: 25 mM MES, pH 6.0
• Recombinant Human Sialyltransferase 6/ST6GALNAC6 (rhST6GALNAC6) (Catalog # 7425-GT)
• CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
• Fetuin (Sigma, Catalog # F3385), 50 mg/mL stock in deionized water
• Sialyltransferase Activity Kit (Catalog # EA002)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard 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 nmoles per well.
3. Dilute CMP-Sialic Acid to 2.5 mM in Assay Buffer.
4. Dilute Coupling Phosphatase 2 to 20 µg/mL in Assay Buffer.
5. Prepare reaction mixture by combining 60 µL of 2.5 mM CMP-Sialic Acid, 60 µL of 20 µg/mL Coupling Phosphatase 2, 60 µL of 50 mg/mL Fetuin, and 120 µL of 100 mM MnCl₂ (supplied in kit). (Volume is sufficient for 12 wells.)
6. Dilute rhST6GALNAC to 5 µg/mL in Assay Buffer.
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 5 µg/mL rhST6GALNAC 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 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 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.

• rhST6GALNAC:  0.125 μg
• Coupling Phosphatase 2: 0.1 µg
• CMP-Sialic Acid:  250 µM
• Fetuin: 250 µg
• MnCl₂: 20 mM