Recombinant Drosophila Notch EGF20 Protein, CF

• Assay Buffer: 25 mM Tris, 10 mM MgCl₂, 10 mM CaCl₂,  pH 7.5
• Recombinant Mouse EOGT (rmEOGT) (Catalog # 8116-GT).
• Recombinant Drosophila Notch EGF20 (Notch peptide) (Catalog 8117-DN)
• UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% EtOH
• 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 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 UDP-GlcNac to 10 mM in Assay Buffer.
4. Dilute Coupling Phosphatase I to 20 ng/µL in Assay Buffer.
5. Prepare reaction mixture containing 0.8 mM UDP-GlcNAc, 0.2 mg/mL Notch Peptide and 4 ng/µL Coupling Phosphatase I in Assay Buffer.
6. Dilute rmEOGT to 20 ng/µL 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 20 ng/µL rmEOGT into the plate. Include a Control containing 25 µL of Assay Buffer.
9. Add 25 µL of reaction mixture to the wells, excluding the standard curve and curve blank.
10. Incubate sealed plate at 37 ºC for 4 hours.
11. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix briefly.
12. Add 100 µL of deionized water to all wells. Mix briefly.
13. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
14. Read plate at 620 nm (absorbance) in endpoint mode.
15. Calculate specific activity:

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

Per Reaction:

• rmEOGT: 0.5 µg
• Coupling Phosphatase I: 0.1 µg
• Notch Peptide: 5 µg
• UDP-GlcNAc: 0.4 mM