I cloned my gene of interest in Picz alpha A vector and overexpressed it in P.pastoris X33. When I run SDS PAGE or do western blot I get bigger size protein band than expected which might be due to glycosylation of protein. So I wanted to ask whether I can directly use supernatant (having my expressed protein in it) for deglycosylation or do i need to purify my protein before deglycosyltaion?? And also can I use deglycosylated protein for western blot analysis?? Thanks
Hi there,
Culture supernatant may contain activities which might interfere with deglycosylation. So it would be better to work on purified material.
About WB, it depends on the nature of the epitope detected by the antibody... If you don't know, just compare reactivity of the native and deglycosylated forms of the protein towards the antibody.
I agree with Dominique about deglycosylating in the supernantant. The enzyme may not work properly in that environment.
Do you know if your protein is glycosylated? How much bigger is the protein compared to the expected size? Pichia can add 10 mannose residues to each residue that can be glycosylated which translates into ~2kDa being added to the size of the protein. Also, in my experience, multiple residues are sometimes not glycosylated evenly resulting in different sizes of the protein by ~2kDa. In any case, if you have several or a single species of your protein, deglycosylating should result in a single protein size of what you expect if your protein is indeed glycosylated.
Good Luck!
Hi Tom, Thanks for your reply. My expected protein size is about 32kDa and I get band of approx 45-50 kDa size. Even I read in few other journal articles where size of expected protein was increased by 60kDa due to glycosylation .
The difference is size seems to be a lot for glycosylation and not seeing any intermediate sizes of the protein. But give the deglycosylation a try anyways and see what happens. Be sure to run your protein not deglycosylated and deglycosylated on your gel so you can see the shift down. If it doesn't shift then you have some other modification(s) to your protein.
Good Luck!
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