What type of protein is it? Do you have a recombinant source? If yes, what organism are you using as this will determine what enzymes you can use (e.g, complex mammalian glycans are not cleaved by EndoH, plant/insect derived core fucosylated proteins are not cleaved by PNGaseF etc)

Dear Bram Laukens,

        Thanks for taking the time to answer my query:-)  I would be using CHOLec1 cell lines to express a recombinant protein which expresses only high-mannose glycans. I am aware that both the chosen enzymes (PNGaseF and EndoHF) cleaves high-mannose glycans. My thought was using both the enzymes together (if possible) may cleave my protein of interest better than just having one of the enzymes. But optimizing the use of both enzymes in the same reaction would be a problem and thus I wanted to know if someone has tried it before.

I personally have never used the combination of both enzymes. I do believe the pH optima are also quite different for both enzymes (5.5 for EndoH and around 8 for PNgaseF I believe) so optimizing it will likely be troublesome. Besides, both enzymes cleave differently. In general, EndoH should perform better in deglycosylating native folded proteins  because there is less sterical hindrance for EndoH as it does not have to come 'so close' to the protein compared to PNGaseF. But much depends on the surface topology where the N-glycosylation site is located (accesibility of the N-glycan). In case you have a robust protein, incubating at a higher temperature might help perhaps.

However, in case you are interested to try post-translational 'in vivo' deglycosylation, I can refer you to the paper of my colleagues: http://www.ncbi.nlm.nih.gov/pubmed/24752077. Feel free to contact them!

Hi Nilima. Have you tried periodate oxidation?