• Assay Buffer: 50 mM Tris, 15 mM MgCl₂, pH 7.5
• Recombinant Mouse Carbohydrate Sulfotransferase 5/CHST5 (rmCHST5) (Catalog # 5210-ST)
• 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (PAPS) (Catalog # )
• N-acetyl-alpha -D-glucosamine (GlcNAc)  (Calbiochem, Catalog #1079), 1 M stock in deionized water
• Universal Sulfotransferase Activity Kit (Catalog # )
• 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 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 reaction mixture containing 0.4 mM PAPS, 600 mM GlcNAc, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
4. Dilute rmCHST5 to 4 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 4 µg/mL rmCHST5 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.

• rmCHST5: 0.1 μg
• Coupling Phosphatase 3: 0.5 μg
• PAPS: 0.2 mM
• GlcNAc: 300 mM