Recombinant Human SULT1A3 Protein, CF

• Assay Buffer: 50 mM Tris, 15 mM MgCl₂, pH 7.5
• Recombinant Human Sulfotransferase 1A3/SULT1A3 (rhSULT1A3) (Catalog # 5829-ST)
• 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (Catalog # ES019)
• 1-Naphthol (Sigma, Catalog # N1000), 10 mM 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 provided by the Universal Sulfotransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
2. Prepare a 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 reaction mixture containing 0.4 mM PAPS, 0.4 mM 1-Naphthol, and 0.02 mg/mL Coupling Phosphatase 3 in Assay Buffer.
4. Dilute rhSULT1A3 to 10 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 diluted rhSULT1A3 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
7. Add 25 µL of reaction 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. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
10. Add 100 µL of deionized water to all wells. Mix briefly.
11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
12. Read plate at 620 nm (absorbance) in endpoint mode.
13. Calculate specific activity:

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

Per Reaction:

• rhSULT1A3: 0.25 µg
• PAPS: 10,000 pmol (0.2 mM)
• 1-Naphthol: 0.2 mM
• Coupling Phosphatase 3: 0.5 µg