Recombinant Human NDST1 Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -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 human N‑Deacetylase/N‑Sulfotransferase 1/NDST1 (rhNDST1) (Catalog # 5967-ST)
• PAPS (3'-Phosphoadenosine-5'-Phosphosulfate) (Catalog # ES019)
• 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 diluting 10X stock 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 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 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 PAPS, 2 mg/mL Heparin Sulfate, and 20 µg/mL Coupling Phosphatase 3 in 1X Assay Buffer.
6. Dilute rhNDST1 to 2 µg/mL in 1X Assay Buffer.
7. Load 25 µL of the 2 µg/mL rhNDST1 into empty wells of the same plate as the curve. Include a control containing 25 µL of Assay Buffer.
8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
9. Seal plate 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 fitting and adjusted for Control.

• rhNDST1: 0.05 µg
• Coupling Phosphatase 3: 0.5 µg
• Heparan Sulfate: 50 ug
• PAPS: 0.2 mM