1. Combine 1-20 µg of glycoprotein, 1 µl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 µl total reaction volume.
2. Denature glycoprotein by heating reaction at 100°C for 10 minutes.
3. Make a total reaction volume of 20 µl by adding 2 µl 10X GlycoBuffer 2, 2 µl 10% NP40, H2O and 1-2 µl PNGaseF.
4. Incubate reaction at 37°C for 1 hour.
Note: We recommend limiting PNGaseF 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 PNGaseF and larger reaction volumes.