Recombinant Human CHST3 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, 50 mM MgCl₂, pH 7.5
• Recombinant Human Carbohydrate Sulfotransferase 3/CHST3 (rhCHST3) (Catalog # 7450-ST)
• Universal Sulfotransferase Activity Kit (Catalog # EA003)
• Donor Substrate: Adenosine 3’-phosphate 5’-phosphosulfate lithium salt hydrate (PAPS) (Catalog # ES019)
• Acceptor Substrate: Chondroitin Sulfate (Sigma, Catalog # C6737), 50 mg/mL stock in deionized water
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute Coupling Phosphatase 3 to 100 μg/mL in Assay Buffer.
2. Prepare a reaction mixture by combining 200 μL 1 mM PAPS, 100 μL 50 mg/mL chondroitin sulfate, and 50 μL 100 μg/mL Coupling Phosphatase 3. This is enough volume for 10 reactions.
3. Dilute rhCHST3 to 8.33 µg/mL in Assay Buffer.
4. 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.
5. 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.
6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
7. Load 15 µL of the 8.33 µg/mL rhCHST3 into the plate. Include a Substrate Blank containing 15 µL of Assay Buffer.
8. Add 35 µL of reaction mixture to the wells, excluding the standard curve.
9. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
11. Add 100 µL of deionized water to all wells. Mix briefly.
12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
13. Read plate at 620 nm (absorbance) in endpoint mode.
14. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

• rhCHST3: 0.125 µg
• Coupling Phosphatase 3: 0.500 μg
• PAPS: 0.4 mM
• Chondrointin Sulfate: 500 μg