Recombinant Human N-Acetylglucosaminyltransferase1/MGAT1, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Buffer A: 25 mM MES, 10 mM MnCl₂, 0.02% Brij-35, pH 6.5
• Buffer B: 100 mM Tris, 5 mM CaCl₂, pH 7.5
• Recombinant Human N-Acetylglucosaminyltransferase I/MGAT1 (rhMGAT1) (Catalog # 8334-GT)
• Donor Substrate: UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
• Acceptor Substrate: alpha 1-3, alpha 1-6 Mannotriose (V-Labs, Catalog # M336), 20 mM stock in deionized water
• Glycosyltransferase Activity Kit (Catalog # EA001)
• 96-well Clear Plate (Catalog # DY990)
• 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 rhMGAT1 to 20 µg/mL in Buffer A.
4. Create Substrate Mixture containing 0.4 mM UDP-GlcNAc and 2 mM alpha 1-3, alpha 1-6 Mannotriose in Buffer A.
5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Buffer A.
6. Load 25 µL of the 20 µg/mL rhMGAT1 into the plate. Include a control containing 25 µL of Buffer A.
7. Start the reaction by adding 25 µL of Substrate Mixture to the wells, excluding the standard curve.
8. Cover the plate with a plate sealer and incubate at 37 °C for 1 hour.
9. Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
10. Add 50 µL of 2 µg/mL Coupling Phosphatase 1 to reaction wells and controls, excluding the standard curve. Also, add 50 µL of Buffer B to the wells containing the standard curve.
11. Mix and incubate for 10 minutes at room temperature.
12. Add 30 µL of the Malachite Green Reagent A to all wells.
13. Add 50 µL of deionized water to all wells.
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.

• rhMGAT1: 0.5 µg
• Coupling Phosphatase 1: 0.1 µg
• UDP-GlcNAc: 0.2 mM
• alpha 1-3, alpha 1-6 Mannotriose: 1 mM