FAQ: I tried the Protein Deglycosylation Mix II on my glycoprotein and didn't see removal of the carbohydrate. What could be the problem?

The Protein Deglycosylation Mix II includes the enzymes necessary to remove carbohydrates attached to Asparagine residues, simple core 1 and core 3 O-linked carbohydrates attached to Ser/Thr, and decorated core 1 and 3 O-glycans (with lactosamine extensions).  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 α1-3Fucose (often found in plant or insect proteins). Also, O-glycans extended with fucose, alpha-galactose, and other residues will be resistant to this cocktail, unless additional exoglycosidase enzymes are added.

If possible, use 10X Deglycosylation Mix Buffer 2 to reduce the protein prior to deglycosylation (this buffer is compatible with sample preparation for mass spectrometry). The secondary and tertiary structure of proteins can prevent endoglycosidases from reaching their substrate, thus making reduction a crucial step in efficient cleavage. If you do not want to reduce then consider adding more enzyme and using longer incubation times.

The sample itself could cause enzyme inactivation. Avoid sample buffers containing SDS as this detergent inhibits PNGase F, O-glycosidase, and β1-4 Galactosidase. The sample could also cause a drop (or rise) in pH, particularly if large volumes are used (in those cases, it would be ideal to exchange the sample in a low molarity, neutral buffer).

Confirm that your target protein should be glycosylated. Some glycosylation predictors might annotate sites that are not occupied in nature. Also, glycosylation patterns change among different tissues, organisms, and/or growth stages. Likewise, different expression systems might result in proteins with unexpected changes in glycosylation.

Finally, the shift in mobility of your protein (SDS-PAGE, Western blot) may be difficult to visualize (particularly when a small carbohydrate has been removed from a large protein). If possible run side by side negative controls. Optimize loading and running conditions to facilitate the detection of subtle mass changes.