Recombinant Human SULT2A1 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 (supplied in 10X form in kit)
• Recombinant Human Cytosolic Sulfotransferase 2A1/SULT2A1 (rhSULT2A1) (Catalog # 5828-ST)
• 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
• T-Dehydroandrosterone (DHEA)  (Sigma, Catalog # D4000), 1 mM in 50% DMSO, 50% deionized water
• Universal Sulfotransferase Activity Kit (Catalog # EA003)
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute Coupling Phosphatase 3 to 0.1 mg/mL in Assay Buffer.
2. Prepare reaction mixture by combining 140 µL of 1 mM PAPS, 70 µL of 1 mM DHEA, 70 µL of 0.1 mg/mL Coupling Phosphatase 3, and 70 µL of Assay Buffer.
3. Dilute rhSULT2A1 to 40 µg/mL in Assay Buffer.
4. 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.
5. 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.
6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
7. Load 25 µL of the 40 µg/mL rhSULT2A1 into the plate. Include a Control containing 25 µL of Assay Buffer.
8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
9. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
11. Add 100 µL of deionized water to all wells. Mix briefly.
12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
13. Read plate at 620 nm (absorbance) in endpoint mode.
14. Calculate specific activity:

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

• rhSULT2A1: 1.0 µg
• PAPS: 10000 pmoles (0.2 mM)
• DHEA: 0.1 mM
• Coupling Phosphatase 3: 0.5 µg