HomeFAQsI tried deglycosylating my glycoprotein with Remove-iT® PNGase F but did not see removal of the carbohydrate. What could be the problem?
FAQ: I tried deglycosylating my glycoprotein with Remove-iT® PNGase F but did not 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)? Remove-iT® 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 not seeing deglycosylation and you know your protein has N-glycans consider adding more enzyme and using longer incubation times. If you have chosen to denature the glycoprotein, be certain to add NP-40 (to protect PNGase F, Recombinant from the SDS in the denaturation step). Outside these reaction conditions there is the possibility that the carbohydrate may be resistant to PNGase F, Recombinant (a rare occurrence). This happens when the core N-acetylglucosamine is modified by an α1-3Fucose (often found in plant and insect proteins).