SULT1A1 Protein, Human (His)

Materials
Test protein: SULT1A1 Protein, Human (His) (HY-P70969)
1-Naphthol, 10 mM in ethanol
3'-Phosphoadenosine-5'-phosphosulfate (PAPS)
Universal Sulfotransferase Activity Kit, containing Assay Buffer (50 mM Tris, 15 mM MgCl₂, pH 7.5)

Procedure
1. Dilute the 10× buffer from the kit to 1× buffer.
2. Dilute the phosphate standard to concentrations of 5000, 2500, 1250, 625, 313, 156, 78, and 0 pmol.
3. Prepare a reaction mixture containing 0.4 mM PAPS, 0.4 mM 1-naphthol, and 0.02 mg/mL coupled phosphatase 3 using 1× buffer.
4. Dilute the test protein to 20 μg/mL with 1× buffer.
5. Add 50 μL of the standard curve dilutions to each well of a 96-well plate, and add 50 μL of 1× buffer to the blank wells.
6. Add 25 μL of Human SULT1A1 to the appropriate wells, and add 25 μL of 1× buffer to the blank wells.
7. Add 25 μL of the reaction mixture to all wells except those used for the standard curve.
8. Seal the plate and incubate at 37 °C for 20 minutes.
9. Add 30 μL of Malachite Green Reagent A to all wells and mix.
10. Add 100 μL of deionized water to all wells and mix.
11. Add 30 μL of Malachite Green Reagent B to all wells, mix, and incubate at room temperature for 20 minutes.
12. Read the absorbance at 620 nm using a microplate reader.
13. Calculate the specific activity: