Recombinant Human GALNT3 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: 25 mM Tris, 0.15 M NaCl, 5 mM MnCl₂ (supplied in kit), 2.5 mM CaCl₂, pH 7.5
• Recombinant Human Polypeptide GalNac Transferase 3/GALNT3 (rhGALNT3) (Catalog # 7174-GT)
• UDP-GalNAc (Sigma, Catalog # U5252), 10 mM stock in deionized water
• EA2 peptide (AnaSpec Inc, Catalog # 63841), 5 mM in 5 mM Tris, pH 7.0
• Glycosyltransferase Activity Kit (R&D Systems, Catalog# EA001)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
2. Prepare 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.
3. Dilute EA2 peptide to 2 mM in Assay Buffer.
4. Dilute Coupling Phosphatase I to 60 ng/µL in Assay Buffer.
5. Prepare reaction mixture by combining 35 μL of 10 mM UDP-GalNAc, 87.5 μL of 2 mM EA2 peptide, 46.7 μL of 60 ng/μL Coupling Phosphatase I, and 180.8 μL Assay Buffer (sufficient to test 13 wells).
6. Dilute rhGALNT3 to 4 ng/µL in Assay Buffer.
7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
8. Load 25 µL of the 4 ng/µL rhGALNT3 into the plate. Include a Control containing 25 µL of Assay Buffer.
9. Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
10. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
12. Add 100 µL of deionized water to all wells. Mix briefly.
13. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
14. Read plate at 620 nm (absorbance) in endpoint mode.
15. Calculate specific activity:

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

• rhGALNT3: 0.1 µg
• Coupling Phosphatase I: 0.2 µg
• EA2 peptide: 0.25 mM
• UDP-GalNAc: 0.5 mM