Please, If anybody could help, we are producing r-IgT from fish in mammalian system and we got a smear on SDS-PAGE, we send parts of gel smear to the MS, but unfortunately results are quite faraway from what we cloned and produced, do we need any special treatment for our glycoprotein (r-IgT) before Ms.
See this page:
https://www.researchgate.net/post/Why_do_glycosylated_proteins_appear_as_a_smear_on_SDS-PAGE2
I hope it may help
Regards
Jose
Hi, Haitham! Let me see if I get your point... If you want to get rid of the glycosylations before you perform the MS in order to evaluate only the protein part of your sample, you could/should treat your samples with either PNGase F or Endo-H, two non-specific glycosidades that wil remove most of, if not all, the glycosylation. Thus you will have a clean, deglycosylated protein sample for the MS run.
You did not mention how you stained the SDS-PAGE, but I assume you used Coomassie Blue. If so, you are staining proteins, but do not identify whether all of them are glycosylated or not. One very simple stain for glycoproteins in SDS-PAGE is as follows: Fix the gel contents by incubation 1-2h (or overnight) with a 30% ethanol/ 10% acetic acid solution (the gel could shrink a bit). Rinse with distilled water, and incubate the gel 15-30 min with a 1% periodic acid or potassium periodate solution (as oxidant), rinse again with water, and thereafter stain glycoproteins (glycosylated proteins in your case) with a simple incubation with Schiff reagent (acid fucsin, will react with oxidized sugars), for a certain time (can vary from 10 to 30 min), followed again by gel washing with water. As the background faints, you will be able to spot the stained pink-to-red glycoprotein bands against a light surface or on a basic transilluminator. Some protocols use a NaOH wash to improve the background destaining, but it is not mandatory, and sometimes even your bands will get too faint to see them on a basic pH, and then you'll need to re-acidify your gel with 0.01-0.1 M HCl... I think you can get a fine protocol for this staining in Sasse & Gallagher, 2009.(link below)
Following this procedure, you will be able to see if glycosylated bands co-localize with your Coomassie blue bands, and if your smear is due to glycosylation, or if you have a glycosylation ladder.
Remember that, if you perform deglycosylation with PNGase F or Endo H before the SDS-PAGE, the protein bands will be shifted downwards, since they will be lighter and with smaller hydrodynamic radius, and should not stain with fucsin, but only with Coomassie blue. I append some links that might be useful. Best wishes, and good luck!
http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb1006s85/pdf
http://www.sigmaaldrich.com/life-science/proteomics/post-translational-analysis/glycosylation/glycoprotein-analysis-manual.html
http://www.dako.com/08066_12may10_webchapter19.pdf
http://www.hindawi.com/journals/ijcc/2012/640923/
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