We want to compare the glycosylated and deglycosylated forms of the target protein on a blot. Tell me how to understand what form of glycosylation a particular protein has? I can’t find reagents for deglycosylation because I don’t know what type I have.
Hi,
Generally speaking, proteins can be glycosylated on asparagines (also referred to as N-linked glycosylation), or on serines and threonines (also referred to as O-linked glycosylation).
The first can be best studied using enzymatic release, for example with PNGase F, or Endo H and the latter chemically, using hydrolysis.
N-linked glycosylation usually deals with larger structures (glycans) that those O-linked, so the (enzymatic) removal of N-linked glycans might be easier to spot on a gel/blot.
Hope this helps!
Dear Дарья Коломийцева
To characterize well the glycosilation pattern of a protein you need to submit it to mass spectrometry characterization:
eg:
https://covalx.com/services/glycan-analysis/
in absence of this kind of analisys you can try to have some more rude information or by incubating your protein with different enzimes with different specificity (e.g pNGase, endoH, o-glycosidase)
and see whats happen in SDS-page
try to perfrom a sort of ELISA by staining your protein with lectins (conjugated with HRP) with different specificity, that can in some way give you an idea of kind of glicosilation
https://en.wikipedia.org/wiki/Lectin
good luck
Manuele
You can deglycosylate using PNgase F to check the protein carries N -glycosyaltion or not. if it has N glycans there will be reduction in molecular weight. Further to characterize type and site you can do Mass spectometry.
Best Wishes
Amit
Amit Ranjan Manuele Martinelli Eef Dirksen Colleagues, thank you very much! You helped me a lot!
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