HomeFAQsI tried the Protein Deglycosylation Mix on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
FAQ: I tried the Protein Deglycosylation Mix on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?
The Protein Deglycosylation Mix includes the enzymes necessary to remove carbohydrates attached to Asparagine residues, simple core 1 and core 3 O-linked carbohydrates attached to Sereine/Threonine, and some complex O-linked glycans. There is the possibility that the carbohydrate may be resistant to PNGase F (a rare occurrence). This happens when the core N-Acetylglucosamine is modified by an alpha1-3Fucose (often found in plant proteins). 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 using longer incubation times. If you have denatured the protein, be certain to add 10% NP-40 (to protect PNGase F and O-Glycosidase from the SDS in the denaturation step).