Recombinant Human SULT2B1 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 2B1/SULT2B1 (rhSULT2B1) (Catalog # 6174-ST)
• Pregnenolone (Sigma, Catalog # P9129), 50 mM stock in DMSO
• Dimethylsulfoxide (DMSO)
• 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
• 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. Dilute pregnenolone to 5 mM in DMSO.
3. Dilute PAPS to 1 mM in Assay Buffer.
4. Prepare reaction mixture by combining 50 µL of 0.1 mg/mL Coupling Phosphatase 3, 20 µL of 5 mM pregnenolone, 100 µL of 1 mM PAPS, and 80 µL of Assay Buffer. This is sufficient to assay 9 wells.
5. Dilute rhSULT2B1 to 80 µg/mL in Assay Buffer.
6. Dilute the 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 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 80 µg/mL rhSULT2B1 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. Seal plate and incubate at 37 °C for 30 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.