Recombinant Mouse CHST3 Protein, CF

• Assay Buffer: 50 mM Tris, 125 mM NaCl, 50 mM MgCl₂, pH 7.5
• Recombinant Mouse Carbohydrate Sulfotransferase 3/CHST3 (rmCHST3) (Catalog # 5356-ST)
• Donor Substrate: Adenosine 3’-phosphate 5’-phosphosulfate (PAPS) (Catalog # ES019)
• Acceptor Substrate: Chondroitin Sulfate (Sigma, Catalog # C6737), 50 mg/mL 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 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 0.571 mM PAPS, 14.3 mg/mL chondroitin sulfate, and 14.3 μg/mL Coupling Phosphatase 3.
4. Dilute rmCHST3 to 4.17 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 15 µL of the 4.17 µg/mL rmCHST3 into the plate. Include a Control containing 15 µL of Assay Buffer.
7. Add 35 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:

• rmCHST3: 0.0625 μg
• Coupling Phosphatase 3: 0.500 μg
• PAPS: 0.4 mM
• Chondrointin Sulfate: 500 μg