Hi Ana,

You can use PNGase F (from NEB or Sigma) to deglycosylate your proteins. Even though it is recommended to denaturate proteins at 100oC before to apply PNGase F, the enzyme is sufficiently active to deglucosylate your proteins even though they are not denaturated. Keep in mind that PNGase only removes N-glycans and will not affect O-glycans. 

Hope this helps. Norbert

You can please follow the links below to obtain certain valuable infomations:-

1. http://www.sigmaaldrich.com/life-science/proteomics/post-translational-analysis/phosphorylation/protein-deglycosylation.html

2. https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/avidin-biotin-complex-method-ihc-detection.html

3. https://www.neb.com/protocols/1/01/01/reaction-protocol-for-protein-deglycosylation-mix-p6039

Thank you both for your useful and fast answers. I will try it!

Ana

Dear all, 

I want use PNGase F to treat my protein, but my question is if I can use that in a total protein extract or if I have to purify the glycoprotein.  Do you have a detailed protocol?

thanks in advance

Francesca

Dear Francesca,

I used the Deglycosylation Mix (P6039) from New England BioLabs and I followed manufacturer instructions. I did it in the total protein extract.

Good luck!

Ana

Dear Ana,

How much did you use total protein as ug? 

Thank you.

Dear Harun,

I used 100 ug of total protein for the deglycosylation.

Could somebody probably refer to dePEGylation protocol for protein in vitro studies?

Thanks in advance,

Victor