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anion exchange chromatography for protein fractionation

Posted: Mon Mar 23, 2015 12:11 am
by cavaliersun2003
Hi all,
I'm wondering if anyone here have tested anion exchange chromatography for protein fractionation before trypsin digestion instead of using 1Dgel.
I have came across protein fractionation protocol using C18, C4 columns before trypsin digestion, but my sample have SDS, so I'm wondering if ion exchange can help solve the problem.
thanks!

Posted: Mon Mar 23, 2015 2:17 am
by zougman
Dear cavaliersun2003,

It is not feasible to perform any type of ion-exchange fractionation on an SDS-solubilized protein sample. You can, however, perform the true size exclusion chromatography (SEC) in this case as was practically demonstrated by Wisniewski et al. http://www.ncbi.nlm.nih.gov/pubmed/20415495

Hope this helps,

AZ

Posted: Mon Mar 23, 2015 2:19 pm
by AAlpert
HILIC gets rid of SDS and other surfactants from proteins and peptides quite nicely. What are your proteins? Will they tolerate a predominantly organic solvent?

An alternative is to try anion-exchange with a lot of organic solvent present, although not as much as is necessary for retention in pure HILIC. Say, 40%.

Posted: Mon Mar 23, 2015 5:38 pm
by cavaliersun2003
Thank you Alpert, I am not interested in a particular protein, but the whole proteome in general. I guess adding organic solvent will cause protein precipitation so I tend to avoid that.
Question to Zougman, I think SDS can be removed by ion-exchange, am I wrong?

Posted: Tue Mar 24, 2015 2:49 pm
by zougman
Hi cavaliersun2003,

The portion of SDS not bound to proteins can be removed by the anion-exchange from the SDS-protein mixture at the acidic pH -- you are right.

AZ

Posted: Wed Mar 25, 2015 7:59 am
by Infinity
Well, can you redo your sample prep this time w/o SDS. It could be a more feasible option compared to removing SDS from proteins.