Recombinant Human ST6GALNAC1 Protein

• Sialyltransferase Activity Kit (Catalog # EA002)
• 10X Assay Buffer (supplied in kit): 250 mM Tris, pH 7.5
• Recombinant Human ST6 Sialyltransferase 1/ST6GALNAC1 (rhST6GALNAC1) (Catalog # 9154-GT)
• CMP-Sialic acid (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. Prepare 1X Assay Buffer containing 10 mM MnCl2 by combining 10X stocks and diluting 10 fold with deionized water.
2. 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 1X Assay Buffer for a 100 µM stock.
   
3. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
   
4. Prepare reaction mixture containing 0.5 mM CMP-Sialic Acid, 20 mg/mL Fetuin and 4 µg/mL Coupling Phosphatase 2 in 1X Assay Buffer.
5. Dilute rhST6GALNAC1 to 20 µg/mL in 1X Assay Buffer.
6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
7. Load 25 µL of the 20 µg/mL rhST6GALNAC1 into the plate. Include a control containing 25 µL of 1X Assay Buffer.
8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
9. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
11. Add 100 µL of deionized water to all wells. Mix briefly.
12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
13. Read plate at 620 nm (absorbance) in endpoint mode.
14. Calculate specific activity:

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

Per Reaction:

• rhST6GALNAC1: 0.5 µg
• CMP-Sialic Acid: 250 µM
• Fetuin: 500 µg
• Coupling Phosphatase 2: 0.1 µg