Recombinant Human ST8SIA2 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.

• Assay Buffer: 50 mM MES, 5 mM MgCl₂, pH 7.0
• Recombinant Human alpha -2,8‑Sialyltransferase 8B/ST8SIA2 (rhST8SIA2) (Catalog # 6590-GT)
• Donor Substrate: CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
• Acceptor Substrate: Recombinant Human NCAM-1/CD56 (rhCD56) 120 isoform (Catalog # 2408-NC)
• 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 3 mM in Assay Buffer.
2. Dilute Coupling Phosphatase 2 to 24 µg/mL in Assay Buffer.
3. Dilute rhCD56 to 240 µg/mL in Assay Buffer.
4. Prepare reaction mixture by combining 45 µL of 3 mM CMP-Sialic Acid, 45 µL of 24 µg/mL Coupling Phosphatase 2, and 180 µL of 240 µg/mL rhCD56.
5. Dilute rhST8SIA2 to 10 µg/mL in Assay Buffer.
6. 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. This is the first point of the standard curve.
7. 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.
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 rhST8SIA2 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.

• rhST8SIA2: 0.250 μg
• Coupling Phosphatase 2: 100 ng
• rhCD56: 4 µg
• CMP-Sialic Acid: 250 µM