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 H2O (if necessary) to make a 10 μl total
- Denature the glycoprotein by heating the reaction
at 55°C for 10 minutes.
- Make a total reaction volume of 20 μl by adding
2 μl 10X GlycoBuffer 2, H2O and 1–5 μl
Endo D .
- Incubate the reaction at 37°C for 1 hour.
Note: To deglycosylate various glycoproteins under native conditions, longer incubation times as well as more enzyme may be required. Reactions may be scaled-up linearly to accommodate larger reaction volumes.