FAQ: I tried the PNGase F on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?

Do you know how the carbohydrate is attached to the protein? Is it N-, or O-linked (linked to an Asn, or a Ser/Thr)? PNGase F will only remove carbohydrates attached to an Asn. NEB’s O-Glycosidase (NEB# P0733) will remove core 1 and core 3 O-linked carbohydrates attached to Ser/Thr. If you are certain you have N-linked carbohydrates, make sure that you denature the protein prior to deglycosylation. The secondary and tertiary structure of proteins can prevent endoglycosidases from reaching their substrate, thus making denaturation a crucial step in efficient cleavage. If you do not want to denature then consider adding more enzyme and longer incubation times. If you have denatured the protein, be certain to add NP-40 (to protect PNGase F from the SDS in the denaturation step). Outside these reaction conditions there is the possibility that the carbohydrate may be resistant to PNGase F (a rare occurrence). This happens when the core N-acetyl-glucosamine is modified by an α1-3Fucose (often found in plant proteins).