Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.
- Combine 1–20 μg of glycoprotein, 1 μl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 μl total reaction volume.
- Denature glycoprotein by heating reaction at 100°C for 10 minutes.
- Make a total reaction volume of 20 μl by adding 2 μl 10X GlycoBuffer 2, 2 μl 10% NP40, H2O and 1–5 μl PNGase F, Recombinant.
- Incubate reaction at 37°C for 1 hour.