Recombinant Human CHST4 Protein, CF

• Assay Buffer (1X Phosphatase Buffer 3): 50 mM Tris, 15 mM MgCl₂, pH 7.5
• Recombinant Human Carbohydrate Sulfotransferase 4/CHST4 (rhCHST4) (Catalog # 5357-ST)
• 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
• N-acetyl-alpha -D-glucosamine (GlcNAc)  (Calbiochem, Catalog # 1079), 1 M stock 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 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.
2. 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.
3. Prepare a reaction mixture containing 0.4 mM PAPS, 20 mM GlcNAc and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
4. Dilute rhCHST4 to 10 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 10 µg/mL rhCHST4 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.

Per Reaction:

• rhCHST4: 0.25 µg
• Coupling Phosphatase 3: 0.500 μg
• PAPS: 0.2 mM
• GlcNAc: 10 mM