Recombinant Human SULT1A1 Protein, CF

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

• Assay Buffer: 50 mM Tris, 15 mM MgCl₂, pH 7.5 (1X Phosphatase Buffer 3)
• Recombinant Human Cytosolic Sulfotransferase 1A1/SULT1A1 (rhSULT1A1) (Catalog # 5546-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 by combining 40 µL with 360 µL of Assay Buffer for a 100 µM stock. This is the first standard curve point.
2. Prepare additional points 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 rhSULT1A1 to 20 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 20 µg/mL rhSULT1A1 into the plate. Include a Control 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 Control.

• rhSULT1A1: 0.5 µg
• PAPS: 10,000 pmol (0.2 mM)
• 1-Naphthol: 0.2 mM
• Coupling Phosphatase 3: 0.5 µg