Recombinant Human GALNT13 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.

• Glycosyltransferase Activity Kit (Catalog # EA001)
• 10X Assay Buffer (supplied in kit): 250 mM Tris, 100 mM CaCl₂, pH 7.5
• MnCl₂ (supplied in kit): 100 mM
• Recombinant Human Polypeptide GalNAc Transferase 13/GALNT13 (rhGALNT13) (Catalog # 8906-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
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare 1X Assay Buffer containing 10 mM MnCl₂ by combining 10X stocks and diluting 10 fold with deionized water.
2. Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Kit 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. Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
4. Prepare reaction mixture containing 1 mM UDP-GalNAc, 0.4 mM EA2 peptide, and 4 µg/mL Coupling Phosphatase 1 in 1X Assay Buffer.
5. Dilute rhGALNT13 to 4 µg/mL in 1X Assay Buffer.
6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
7. Load 25 µL of 4 µg/mL rhGALNT13 into empty wells of the same plate as the curve. Include a Control containing 25 μL of 1X Assay Buffer.
8. Add 25 µL of the reaction mixture to all 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 sealed plate 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.

• rhGALNT13: 0.1 µg
• Coupling Phosphatase 1: 0.1 µg
• UDP-GalNAc: 0.5 mM
• EA2 peptide: 0.2 mM