Recombinant Human N-Acetylglucosaminyltransferase V/MGAT5 CF

• Buffer A: 25 mM MES, pH 6.0
• Buffer B: 100 mM Tris, 5 mM CaCl₂, pH 7.5
• Recombinant Human N‑Acetylglucosaminyltransferase V/MGAT5 (rhMGAT5) (Catalog # 5469-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 Donor Substrate to 380 μM in Buffer A.
2. Dilute Acceptor Substrate to 4 mM in Buffer A.
3. Prepare Substrate Mixture by combining equal volumes of 380 μM Donor Substrate and 4 mM Acceptor Substrate.
4. Dilute rhMGAT5 to 40 µg/mL in Assay Buffer.
5. Dilute 1 mM Phosphate Standard by adding 40 µL of the Phosphate Standard to 360 µL of deionized water for a 100 µM stock.
6. Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Buffer A. The standard curve has a range of 0.047 to 3 nmol per well.
7. Load 60 µL of each dilution of the standard curve into a plate. Include a curve blank containing 60 μL of Buffer A.
8. Load 20 µL of the 40 µg/mL rhMGAT5 into the plate. Include a substrate blank containing 20 µL of Buffer A.
9. Start the primary reaction by adding 20 µL of Substrate Mixture to the wells, excluding the standard curve.
10. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 1 hour.
11. Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
12. Add 20 µL of 2 µg/mL Coupling Phosphatase 1 to reaction wells and blanks, excluding the standard curve  (this is the secondary or coupling reaction).
13. Mix and incubate for 10 minutes at room temperature.
14. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
15. Add 90 µL deionized water to all wells.
16. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
17. Read plate at 620 nm (absorbance) in endpoint mode.
18. Calculate specific activity:

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

      **Based upon a 60 minute incubation time.

Per Reaction:

• rhMGAT5: 0.8 µg
• Coupling Phosphatase 1: 40 ng
• Donor Substrate: 95 µM
• Acceptor Substrate: 1 mM