Optimal incubation times
and enzyme concentrations must be determined
empirically for a particular substrate. Typical
reaction conditions are as follows:
- Combine 10–20 µg of glycoprotein, 1 µl of 10X
DTT and H20 (if necessary, to make a 10 µl total
- 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.