I would like to perform protein fractionation similar to the results of SDS-PAGE, but using an LC system and SEC column. Has anyone ever done smth similar and visualised fractions on a gel?
It is OK to do gel filtration chromatography under denaturing conditions, such as 6 M urea or guanidine HCl. Just check that the resin you are using and the LC system are compatible with these substances.
https://www.jbc.org/article/S0021-9258(18)62832-7/pdf
I have experience running size exclusion chromatography on an analytical HPLC system using 6 M urea in the mobile phase, although we weren't analyzing protein.
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Because of the high solute content, be careful not to let the elution buffer dry up in the pumps, because it will crystallize. The pumps may not be damaged, but it will be troublesome to clean them out. Also, be careful not to get these strongly denaturing solutions on your skin or in your eyes.
You do not have to denature your proteins for fractionation at the SEC-LC system. Is an alternative way of PAGE to roughly fractionate your proteins by sizes. If you combine with MALLS you may also know the MWs of proteins. On the other hand, you may denature your proteins by chaotropic agents but also by reducing them using DTT/TCEP (you indicated as similar to SDS-PAGE) and can inject the resulting solution into the SEC column for denatured fractionation...I guess refolding won't be a problem if a low amount of urea is present in the LC phase. maybe this can also be excluded...
Hellow Pavel V Shliaha
I came across this document, which performed an SEC with different concentrations of SDS (as the denaturing agent) in the mobile phase. Hope it gives you some idea about this.
https://www.agilent.com/cs/library/applications/5991-7989EN.pdf
Also, you need to check the column resin material and its tolerance to denaturants in the mobile phase. Because these denaturants can affect the system. Make sure you wash the column and the pumps adequately after use. Even though it's possible, it's not typically recommended to use denaturing agents in the mobile phase for SEC.
Best wishes!
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