Recombinant Human GAL3ST2 Protein, CF

• Assay Buffer: 25 mM MES, 15 mM MgCl₂, pH 5.5
• Coupling Phosphatase Buffer: 100 mM Tris, 15 mM MgCl₂, pH 8.0
• Recombinant Human Galactose‑3‑O‑sulfotransferase 2/GAL3ST2 (rhGAL3ST2) (Catalog # 7719-ST)
• Donor Substrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
• Acceptor Substrate: alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock in deionized water
• Universal Sulfotransferase Activity Kit  (Catalog # EA003)
• 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. Prepare a Substrate mixture of 0.8 mM PAPS and 10 mM alpha -Lactose in Assay Buffer.
4. Dilute rhGAL3ST2 to 4 µg/mL in Assay Buffer.
5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
6. Load 25 µL of the 4 µg/mL rhGAL3ST2 into the plate. Include a Control containing 25 µL of Assay Buffer.
7. Add 25 µL of Substrate mixture to the wells, excluding the standard curve.
8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
9. Dilute Coupling Phosphatase 3 (supplied in kit) to 10 µg/mL in Coupling Phosphatase Buffer.
10. Add 50 µL of 10 µg/mL Coupling Phosphatase 3 to each well, excluding the standard curve.  Add Coupling Phosphatase Buffer to the standard curve.
11. Cover the plate with a plate sealer and incubate at 37 °C for 10 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix briefly.
13. Add 50 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.
     **Use the incubation time of the reaction between the Enzyme and Substrate mixture only, exclude additional incubation times.

Per Reaction:

• rhGAL3ST2:  0.1 µg
• Coupling Phosphatase 3: 0.5 µg
• PAPS: 20 nmol
• alpha -Lactose: 250 nmol