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Can protein isomers be detected in SDS-PAGE - (Aug/18/2013 )

Hi there


I wanted to isolate a recombinant protein from e.coli of 40 KDa size. After purification I obtained two very close bands in both SDS-PAGE and native page. Both bands react positively to western blot. Also  at 37 degrees the protein was produced in inclusion bodies with the two close bands. 


In all cases the top band is thicker than the bottom one.


Since they are very close together, I am confused if they are isomers or truncated proteins. 



Please let me know if you have something like this in the published papers.






It could be that you have detected isomers, but we need a few more details:


Have you titrated the antibody to ensure that it is specific?

How big are your alterations to the protein size?

What percentage gels are you running and for how long, under what conditions?

Could you do some peptide competition experiments to prove that both bands are specific?

Is it likely (bioinformatically) that one of the bands is from a truncation?


In general, any time you consistently see doublets of your recombinant protein of interest, it's either truncated/proteolyzed protein or a covalently modified form. Cutting out the bands and sending them for MS sequencing is likely to be your most informative route.


Are you boiling your SDS-PAGE samples with mercaptoethanol prior to running on the gel? If you're not boiling, the doublet could be denatured and partially denatured protein. I've seen situations where 2% SDS does not completely denature the protein at room temp. If you're not using BME, you could have an internal disulfide.


If the lower band is truncated protein caused by premature translational terminatio, use a c-terminal affinity tag.


If the truncated protein caused by proteolysis, use a protease inhibitor cocktail during purification and you should get less of the lower band


If none of the prove useful, you likely have some kind of PTM (internal crosslink, lipidation, oxidation, etc). 


Any chance you're expressing your protein with some kind of N-terminal signal sequence on it?  We commonly express proteins that, in the native host, reside in the periplasm and are directed that way via an n-terminal 50 amino acid periplasmic signaling sequence that gets cleaved as it gets transported into the periplasm. When overexpressing these proteins in ecoli, we remove these first 50 or so amino acids from the expression construct otherwise we get strange expression results.


The two replies above are spot on.  Depending on the amount of information on your protein, I use This will tell you all of the experimentally determined post-translational modification sites that are on your protein of interest.  From here you can use inhibitors to see if they alleviate the more prominent band.