Recombinant Human ST8SIA4 Protein, CF

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

• Assay Buffer: 100 mM MES, 10 mM MnCl₂ (supplied in kit), pH 7.0
• Recombinant Human alpha -2,8-Sialyltransferase 4/ST8SIA4 (rhST8SIA4) (Catalog # 7027-GT)
• Donor Substrate: CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
• Acceptor Substrate: Recombinant Human NCAM-1/CD56 120 isoform (rhNCAM-1) (Catalog # 2408-NC)
• Sialyltransferase Activity Kit (R&D Systems, 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 nmol per well.
3. Reconstitute lyophilized rhNCAM-1/CD56 to 500 μg/mL in deionized water, 80 μg is needed for this protocol (do not reconstitute in PBS).
4. Dilute CMP-Sialic Acid to 2.78 mM in Assay Buffer.
5. Dilute Coupling Phosphatase 2 to 22.2 µg/mL in Assay Buffer.
6. Prepare reaction mixture by combining 45 µL of 2.78 mM CMP-Sialic Acid, 45 µL of 22.2 µg/mL Coupling Phosphatase 2, and 160 µL of 500 μg/mL rhNCAM-1/CD56. (Volume is sufficient to test 10 wells.)
7. Dilute rhST8SIA4 to 20 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 20 µg/mL rhST8SIA4 into the plate. Include a Control containing 25 µL of Assay Buffer.
10. Start the reaction by adding 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.

• rhST8SIA4: 0.5 μg
• Coupling Phosphatase 2: 0.1 μg
• rhNCAM-1/CD56: 8 μg
• CMP-Sialic Acid: 0.25 mM