Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF

• Buffer A: 25 mM MES, 5 mM MnCl₂, pH 6.5
• Buffer B: 100 mM Tris, 5 mM CaCl₂, pH 7.5
• Recombinant Human N-Acetylglucosaminyltransferase III/MGAT3 (rhMGAT3) (Catalog # 7359-GT)
• Donor Substrate: UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
• Acceptor Substrate: Biantennary N-Linked Core Pentasaccharide (V-Labs, Catalog # M592), 10 mM 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 1 mM Phosphate Standard provided by the Glycosyltransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Buffer A for a 100 µM stock.
2. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Buffer A. The standard curve has a range of 0.078 to 5 nmol per well.
3. Dilute rhMGAT3 to 10 µg/mL in Buffer A.
4. Dilute Donor Substrate to 1 mM in Buffer A.
5. Dilute Acceptor Substrate to 1 mM in Buffer A.
6. Prepare Substrate Mixture by combining 120 µL of 1 mM Donor Substrate, 120 µL of 1 mM Acceptor Substrate, and 60 µL of Buffer A.
7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Buffer A.
8. Load 25 µL of the 10 µg/mL rhMGAT3 into the plate. Include a Control containing 25 µL of Buffer A.
9. Start the reaction by adding 25 µL of Substrate Mixture to the wells, excluding the standard curve.
10. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
11. Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
12. Add 50 µL of 2 µg/mL Coupling Phosphatase 1 to reaction wells and blanks, excluding the standard curve. Also, add 50 µL of Buffer B to the wells containing the standard curve.  Mix and incubate for 10 minutes at room temperature.
13. Add 30 µL of the Malachite Green Reagent A to all wells.
14. Add 50 µL of deionized water to all wells.
15. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
16. Read plate at 620 nm (absorbance) in endpoint mode.
17. Calculate specific activity:

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

Per Well:

• rhMGAT-3: 0.25 µg
• Coupling Phosphatase 1: 0.1 µg
• Donor Substrate: 10 nmol
• Acceptor Substrate: 10 nmol