Recombinant Human POMGNT1 Protein, CF

• Assay Buffer: 25 mM MES, 5 mM MnCl₂, 2.5 mM CaCl₂, 0.02% Brij-35, pH 6.5
• Recombinant Human POMGNT1 (rhPOMGNT1)  (Catalog # 7414-GT)
• UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
• Methyl-alpha -D-mannopyranoside (Sigma, Catalog # M6882), 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 1 mM Phosphate Standard provided in the kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM Stock.
2. Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
3. Dilute Coupling Phosphatase I to 24 µg/mL in Assay Buffer.
4. Dilute UDP-GlcNAc to 6 mM in Assay Buffer.
5. Prepare Reaction Mixture by combining equal volumes of 24 µg/mL Coupling Phosphatase I, 6 mM UDP-GlcNAc, and 1 M methyl‑ alpha ‑D‑mannopyranoside.
6. Dilute rhPOMGNT1 to 2 µg/mL in Assay Buffer.
7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
8. Load 25 µL of the 2 µg/mL rhPOMGNT1 into the plate. Include a Control containing 25 µL of Assay Buffer.
   Start the reaction by adding 25 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
9. Cover the plate with a plate sealer and incubate at 37 ºC 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 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.  

Per Reaction: