Recombinant Human ST6GALNAC5 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Sialyltransferase Activity Kit (Catalog # EA002)
• Assay Buffer: 25 mM MES, 20 mM MgCl₂, pH 6.5
• Recombinant Human GalNAc alpha -2,6-sialyltransferase V/ST6GALNA (rhST6GALNAC5) (Catalog # 6715-GT)
• CMP-Neu5Ac (Sigma, Catalog # C8271), 10 mM stock in deionized water
• Fetuin (Sigma, Catalog # F3385), 50 mg/mL 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. Dilute CMP-Neu5Ac to 1.5 mM in Assay Buffer.
4. Dilute Fetuin to 30 mg/mL in Assay Buffer.
5. Dilute Coupling Phosphatase 2 to 12 μg/mL in Assay Buffer.
6. Prepare reaction mixture by combining equal volumes of diluted CMP-Neu5Ac, Fetuin, and Coupling Phosphatase 2.
7. Dilute rhST6GALNAC5 to 10 µg/mL in Assay Buffer.
8. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
9. Load 25 µL of the 10 µg/mL rhST6GALNAC5 into the plate. Include a Control containing 25 µL of Assay Buffer.
10. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
11. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
13. Add 100 µL of deionized water to all wells. Mix briefly.
14. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
15. Read plate at 620 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

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

• rhST6GALNAC5: 0.25 μg
• Coupling Phosphatase 2: 100 ng
• CMP-Neu5Ac: 250 µM
• Fetuin: 250 μg