HomeFAQsI tried the Endo H/Hf on my glycoprotein and it failed. What could be the problem?
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.
You have been idle for more than 20 minutes, for your security you have been logged out. Please sign back in to continue your session.
Your profile has been mapped to an Institution, please sign back for your profile updates to be completed.