how to detect differential glycosylation in acrylamide gels

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g a
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how to detect differential glycosylation in acrylamide gels

What is the method to detect the differentially glycosylated isoforms of same protein resolved by SDS tricine PAGE.

We have 3 different MW proteins identified as same using mass spectrometry. My presumption is its differential glycosylation occurs in the three proteins contributing to their variable MW( 14kda, 17kda and 21kDa)

Chin Fen Teo
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 Argerine,

 Argerine,

One thing you can do is to enzymatic remove the potential glycan(s) by a combination of glycosidases and see whether your higher bands get reduce and become a single band. You can use PNGase F (to remove N-glycans) or/and O-glycanase (to remove O-glycans).

Good luck. 

g a
g a's picture
Hi Pippuri

Hi Pippuri

PNGase method I did thought about but It was a quick thought  that I had and I dnt have PNGase as of now... Any other substitution  by chemical methods for the same response???

I did found out that PAS reaction and Alcian blue methods can be used for staining glycoproteins.

Will it be sufficient If we demonstrate the protein identity by MS and then also show the same in a gel being differentially stained using either of the above methods...

Chin Fen Teo
Chin Fen Teo's picture
 Argerine,

 Argerine,

PAS reaction and Alcian blue methods use pretty harsh conditions. Honestly, I am not sure it works on SDS-PAGE (they are mainly use for tissue section, as far as I know)...
In order to directly visualize glycoprotein in a gel, you can try a product from invitrogen, called Pro-Q Emerald glycoprotein gel stain.
I am not aware of chemical method to remove N-glycans, but mild alkaline condition can remove O-glycan via beta-elimination.
To be sure, I would still perform the glycosidase treatments to proof that they are indeed the same protein with distinct glycan modifications...