Recombinant Mouse Heparan Sulfate-6-O-Sulfotransferase-3, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Universal Sulfotransferase Activity Kit (Catalog # EA003)
• 10X Assay Buffer (supplied in kit): 500 mM Tris, 150 mM MgCl₂, pH 7.5
• Recombinant Mouse Heparan Sulfate 6-O-Sulfotransferase 3/HS6ST3 (rhHS6ST3) (Catalog # 5406-ST)
• Donor Substrate: PAP³²S (3'-Phosphoadenosine-5'-Phosphosulfate) (Catalog # ES019)
• Acceptor Substrate: Heparan Sulfate (Celsus Labs, Catalog # HO3105), 50 mg/mL stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare 1X Assay Buffer by combining 10X stock and diluting 10 fold with deionized water.
2. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
3. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
4. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
5. Prepare a reaction mixture containing 0.4 mM PAP³²S and 6 mg/mL Heparan Sulfate in 1X Assay Buffer.
6. Dilute Coupling Phosphatase 3 (supplied in kit) to 50 µg/mL in 1X Assay Buffer.
7. Dilute rmHS6ST3 to 33.34 µg/mL in 1X Assay Buffer.
8. Load 15 µL of the 33.34 µg/mL rmHS6ST3 into empty wells of the same plate as the curve. Include a control containing 15 µL of Assay Buffer.
9. Add 10 µL of 50 µg/mL Coupling Phosphatase 3 to wells containing enzyme and control, excluding the standard curve.
10. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
11. Seal plate and incubate at 37 ºC for 20 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
13. Add 100 µL of deionized water to all wells. Mix briefly.
14. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
15. Read plate at 620 nm (absorbance) in endpoint mode.
16. Calculate specific activity:

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

• rmHS6ST3: 0.5 µg
• Coupling Phosphatase 3: 0.5 µg
• Heparan Sulfate: 150 ug
• PAP³²S: 0.2 mM