FAQ: I tried the Endo H/Hf on my glycoprotein and it failed. What could be the problem?

First, do you know how the carbohydrate is attached to the protein? Is it N-or O-linked (linked to an Asp or Ser/Thr)? Second, is the carbohydrate substrate high mannose type? If you are uncertain a more general endoglycosidase, PNGase F, may be the more appropriate enzyme. If you are certain that there are high mannose, N-linked carbohydrates, make sure that you have denatured the protein prior to deglycosylation. The secondary and tertiary structure of proteins often prevents 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 incubating longer. Remember, Endo H/Hf specifically removes only high mannose and some ""hybrid-type"" carbohydrates from glycoproteins. If the carbohydrate is more complex, then Endo H will not cleave. PNGase F is useful in removing ""all"" types of N-linked carbohydrates from glycoproteins.