Recombinant Human GALNS Protein, CF

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

• Assay Buffer: 50 mM Sodium Actetate, 250 mM NaCl,  pH 5.0
• Recombinant Human N‑Acetylgalactosamine-6-Sulfatase/GALNS (rhGALNS) (Catalog # 8269-SU)
• Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N-7251), 100 mM stock in deionized water
• Sodium Hydroxide (NaOH) (Sigma, Catalog # 221465), 2 M stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute rhGALNS to 40 µg/mL in Assay Buffer.
2. Dilute PNCS to 4 mM in Assay Buffer.
3. Combine 200 µL of 40 µg/mL of rhGALNS and 200 µL of 4 mM of PNCS in microtubes.  Include a Substrate Blank with 200 µL of Assay Buffer and 200 µL of 4 mM of PNCS.
4. Incubate at 37 °C overnight (16-20 hours).
5. Load 100 µL from each reaction and Substrate Blank vial into a plate in triplicate, minimally.
6. Stop reaction by adding 100 µL of 0.2 M NaOH to each well containing reactions and Substrate Blank.
7. Read plate at 510 nm (absorbance) in endpoint mode.
8. Calculate specific activity:

     *Adjusted for Substrate Blank
     ** Derived using calibration standard 4-Nitrocatechol (PNC) (Sigma, Catalog # N15553).

• rhGALNS: 2 µg
• Substrate: 1 mM