Typical reaction conditions are as follows:
- Combine 1-20 μg of glycoprotein, 1 μl of 10X Glycoprotein Denaturing Buffer and H20 (if necessary) to make a 10 μl total reaction volume.
- Denature glycoprotein by heating raection at 100°C for 10 minutes.
- Make a total reaction volume of 20 μl by adding 2 μl of 10X G5 Reaction Buffer, H20 and 1-5 μl Endo H.
- Incubate reaction at 37°C for 1 hour.
- Reactions may be scaled-up linearly to accommodate larger reaction volumes.
- Enzymatic activity is not affected by SDS
- To deglycosylate a native glycoprotein, longer incubation timeas well as more enzyme may be required.
- Activity at different temperatures was measured after a 1 hour incubation of a glycosidase and denatured RNase B at the given temperatures:
37°C - 100%
30°C - 65%
25°C - 40%
17°C - 25%
2°C - 0 %