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 reaction at 100°C for 10 minutes.
- Make a total reaction volume of 20 μl by adding 2 μl of 10X G7 Reaction Buffer, 2 µl of 10% NP-40, H20 and 1-2 μl PNGase F.
- Incubate reaction at 37°C for 1 hour.
- If using P0704, we recommend limiting PNGase F to 1/10 (or less) of the total reaction volume to keep final glycerol concentration equal to (or less than) 5%.
- Reaction may be scaled up linearly to accommodate large amounts of PNGase F and larger reaction volumes.
- Several exo- and endo-glycosidases can be used together in one digest. For buffer recommendations, please reference the Glycosidases Double Digest Chart .
- For a more detailed characterization of several glycosidase enzymes, please reference the Detailed Characterization of Several Glycosidase Enzymes page.
- For unit conversion between different suppliers, please reference the Glycobiology Unit Conversion Chart page.