Hi,
I have two problems, firstly, I need to remove all the glycosylated sugars from my protein of interest, without interrupting the integrity of my protein as I need all its epitopes to remain intact. Secondly I need quite a bit of the final protein, without having to repeat the procedure several times?

Can anyone reccomend a method/kit etc that they know works well?

Thanks
Rachael

Hi
I am not an expert in Glycosilations and DeGlycosilations,
I asked my friend and she told me that there are many EndoF that you can use.
I also found this Word doc. on the web which has alot of protocols of Glycosilations and deglycosilations:
www.biotech.ufl.edu/WorkshopsCourses/ Word_files/GlycoManualfinal4.doc
Try it and let us know.

N-glycosidase F is also known as PNGas F completely removes N-glycans from peptides and proteins

N-glycosidase H is useful for testing the existence of high mannose type and hybrid type asparagine-linked glycan
chains on purified glycoproteins. It cleaves after GlcNAc group immediately attached to the Asn.

Here's a little brochure reference. The Roche kits are pretty good.

http://www.roche-applied-science.com/PROD_INF/BIOCHEMI/No.4_96/p13.pdf

Hi thank you both for your help, my collegue has a kit from sigma that uses TFMS that she said i could have. It completely removes the sugars and its suppose to maintain the protein integrity, i'm going to try it this week and see if it works. i'll let you know how it goes.
thanks again!

Hi i've had ago at deglycosylated my protein, and hit a few problems.
Firstly, once I had chemically treated it with TFMS I desalted the protein by dialysis against PBS, but my protein precipitated and wouldn't go back into solution. Do you have any suggestions for resolubilising my protein? or for preventing this?

To see if I had recoverd any deglycosylated protein I ran a mass spec and this clearly showed that I had no deglycosylated or native glycosylated protein either. I was thinking that it could be a result of the precipitate but also maybe proteases, can you suggest any protease inhibitors I could add, and how I can remove them at a later date?

Kind Regards
Rachael

Hi i've had ago at deglycosylated my protein, and hit a few problems. Firstly, once I had chemically treated it with TFMS I desalted the protein by dialysis against PBS, but my protein precipitated and wouldn't go back into solution. Do you have any suggestions for resolubilising my protein? or for preventing this? To see if I had recoverd any deglycosylated protein I ran a mass spec and this clearly showed that I had no deglycosylated or native glycosylated protein either. I was thinking that it could be a result of the precipitate but also maybe proteases, can you suggest any protease inhibitors I could add, and how I can remove them at a later date? Kind Regards Rachael