Recombinant Human B4GalT1 (Y285L) 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 HEPES, 10 mM MnCl₂ (supplied in kit), pH 7.5
• Recombinant Human beta ‑1,4‑Galactosyltransferase 1/B4GalT1 (Y285L) (rhB4GalT1) (Catalog # 7040-GT)
• Donor Substrate: Uridine 5’-diphospho-N-acetylgalactosamine (UDP-GalNAc) (Sigma, Catalog # U5252), 10 mM stock in deionized water
• Acceptor Substrate: N-Acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog # 1079), 1 M stock in deionized water
• Glycosyltransferase Activity Kit (Catalog # EA001)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute UDP-GalNAc to 1 mM in Assay Buffer.
2. Dilute Coupling Phosphatase 1 to 20 ng/µL in Assay Buffer.
3. Dilute GlcNAc to 200 mM in Assay Buffer.
4. Prepare Reaction Mixture by combining equal volumes of 1 mM UDP-GalNAc, 20 ng/µL Coupling Phosphatase 1, and 200 mM GlcNAc.
5. Dilute rhB4GalT1 to 1 µg/mL in Assay Buffer.
6. 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.
7. 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 5.0 nmol per well.
8. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
9. Load 20 µL of the 1 µg/mL rhB4GalT1 into the plate. Include a Substrate Blank containing 20 µL of Assay Buffer.
10. Start the reaction by adding 30 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
11. Seal plate and incubate at 37 °C for 20 minutes.
12. Add 30 µL of the Malachite Green Reagent A to all wells.
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.

• rhB4GalT1:  0.020 µg
• Coupling Phosphatase 1: 0.2 µg
• UDP-GalNAc: 10 nmol
• GlcNAc: 2000 nmol