It depends on which kind of glycosylation we are dealing with. It could be N- or O- glycosylated. I think most of the glycosydases used to get it off the sugars are more effective with tryptic peptides. Have you check some chemical protocols?

A long shot could be site mutated the glycosation sites. It jus to get rid off the asparagine of the sequon (N-X-S/T) in the case of N-glycosyaltion. However this could have some side effects I the protein folding and expression

Thank you very much for your kind answer. in fact, I have no idea on what kind of glycosylation the tartet protein has. After deglycosylation, the protein will be used as antigen for western blot. therefore, I just expect the linear epitopes are not broken. I found one candidate, TFMS deglycosylation. Do you have any comments on this method?

Hi Xiaoguang,

Why do you want to remove the glycans? Just to make the protein smaller? I would suggest that you go for the N-linked glycans and that you do it enzymatically. I have done this on a 160 kD protein where half of its mass is due its N-linked glycans. 1) Lyse the cells in RIPA buffer if your protein is membrane anchored otherwise milder buffers will do 2) Add protease inhibitors to the lysate, IMPORTANT! 3) Add PNGaseF which is a N-glycanase that makes no discrimination among the N-linked glycans. You can purchase it from New England Biolabs. With their enzyme comes a reducing agent and a buffer, which you add to your lysate to reach the concentrations they suggest. 4) Incubate at 37 °C for 30-60 min. (I have described this in my paper from 2009 if you need to take a closer look.)

My protein survived. I hope yours will too. Good luck!

If you do not denature the protein, you may find that some glycosites are incompletely digested; thus, you may need to perform a separation to obtain the totally deglycosylated protein. Also, during any analysis of the protein, remember that the digestion will convert each glycosylated ASN to ASP.

Yes, what Jeffery says about ASN being converted to ASN will happen if you use PNGaseF. This may change the isoelectric point enough of your protein to make it migrate slightly slower in your SDS-PAGE. However, it shouldn`t affact the immunodetection in WB as the antibodies normally do not arise against peptide sites that are shielded by the glycans...well unless you have made your own antibodies directed to those sites of course. Anyways, you can also deglycosylate with EndoH, but EndoH only cuts off N-glycans with high mannose structure. If your protein happens to be an ER residing protein than you can have a complete deglycosylation with EndoH otherwise most likely not.

I forgot to mention above that once you have added the reducing agent you also need to heat your lysate (100 °C) for 10 min. This is to breake the disulphide bonds if your protein has any, which makes it easier for the N-glycanases access the glycans.

Since you don`t mention why you want to deglycosylate your protein I don`t know if anything of this is relevant to you, but I hope that it will be of some help. :)

Hi, Alenka

I appreciate you very much for your so detailed answers.

I will read the paper you recommended and then may have further question regarding to my experiment or your paper.

My purpose is simple, I just want to get a smaller protein without glycan. thank you very much for your kindess.

Hi, Jeffrey

thank you for your kind answer.

Your words are really helpful for me.

Xiaoguang

Hi again Xiaoguang,

Ok, since you just want to make your protein smaller I think the procedure I described will work for you and also in your case I would choose PNGaseF. EndoH will most likely not be good for your purpose.

No problem, you are welcome to contact me if you have any further questions.

Hi, Alenka

I read your paper (JV 2009) and also checked the PNGase F in NEB. now I understand more about this experiment. Thank you very much for your kind recommendation.

I also find a "Reaction Protocols for Protein Deglycosylation Mix (P6039)" in NEB. the mixture containing PNGase F and some other enzymes. It may help to remove not only N-link but also some O-link. Therefore, I am now considering to use this mixture. If you have any suggestion or comments about it, please let me know. I appreciate you for your kind help on this issue.

Xiaoguang

PS, I have about four years experience on HIV-1 Vaccine/ anti-HIV drug analysis When I was a postdoc in Tohoku University. please let me know if there is anything I can help.

Hi Xiaoguang,

You may not even have to bother about O-linked sugars for the purpose that you have. Have you checked for information about your protein in protein databases? You may find some information there about the glycosylation of your protein.

So with your HIV&HIV drug background my paper was even a little bit within your field? :) Anyways, thank you, I'll keep you in mind if I'll need any advise regarding our common topic in the future and feel free to get in touch with me if you have any further questions. Good luck! - Alenka

Hi, Alenka

I can only find the N-linked sugars reported in my target protein from database, although I am not sure whether some unreported O-links are there. At present, It might be more reasonable to try the PNGase F first. Thank you again for your kind suggestion. I will ask for your help, if I meet new problem on this experiment.

Best wishes

Xiaoguang