Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate.
- Combine 10-20 µg of glycoprotein, 1 µL of 40mM DTT and H20 (if necessary to make a 10 µL total reaction volume.
- Denature glycoprotein by heating reaction at 55°C for 10 minutes.
- Make a total reaction volume of 20 µL by adding 2 µL 10X GlycoBuffer 2, H20 and 1-5 µL Remove-iT® PNGase F.
- Incubate reaction at 37°C for 1 hour.