anion exchange chromatography for protein fractionation

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cavaliersun2003
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anion exchange chromatography for protein fractionation

Postby cavaliersun2003 » Mon Mar 23, 2015 12:11 am

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!

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zougman
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Postby zougman » Mon Mar 23, 2015 2:17 am

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

AAlpert
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Postby AAlpert » Mon Mar 23, 2015 2:19 pm

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%.

cavaliersun2003
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Postby cavaliersun2003 » Mon Mar 23, 2015 5:38 pm

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?

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zougman
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Postby zougman » Tue Mar 24, 2015 2:49 pm

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

Infinity
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Postby Infinity » Wed Mar 25, 2015 7:59 am

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.


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