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Trouble with purified recombinant protein bands


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#1 Bioscientist

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Posted 23 May 2013 - 07:44 PM

Hello everyone

I have expressed a plant protein in E.Coli. This recombinant protein is His-tagged. Its size is 27KDa. Now when I purify my protein and run it in SDS-PAGE I have two bands very close to each other at about 27 KDa. I cut both of them out and sent them for peptide mass fingerprinting. Both of them turned out to be my protein. I have attached a photo of my purified protein in SDS-PAGE. Lane 5, 6, 7 are my purified protein elutions at 100-250mM imidazole. The lower band is lighter than the upper band.

I was wondering why this size difference? It cannot be a dimer as I heat my samples in SDS-loading buffer with DTT. Any suggestions on how is it possible to get the same protein in two close but different sizes, will be highly appreciated. I tried looking this kind of problem in papers but couldn't come across one.

Also I ran a native gel and I got one band at about 73 KDa and another about 65 KDa. They had quite a distance than what they appear in SDS-PAGE.

Bioscientist

Edited by Bioscientist, 23 May 2013 - 07:46 PM.


#2 mdfenko

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Posted 24 May 2013 - 06:26 AM

are there any differences in the mass fingerprints? you may have a slightly truncated version of the protein or different his tag lengths or post-translational modification.

differences of mobility in native page can be due to charge as well as size differences (or a combination).
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#3 Bioscientist

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Posted 24 May 2013 - 09:01 AM

Thanks for the reply.

The only difference is that the upper band has a score of 2216.42 and the lower band has a score of 2082.55. Do you think this slightly truncated version is due to proteases? I thought that if due to protease I would get a lot more smaller peptides in the SDS-PAGE gel. And what are the post-translational modifications possible in E.Coli?

#4 mdfenko

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Posted 24 May 2013 - 10:25 AM

could be proteases. some cleave at the terminus, some have very specific cleavage sites.

there are a number of ptms possible in e coli (eg proteolysis, phosphorylation,...). some are engineered into some strains (eg glycosylation).

phosphorylation could make a protein migrate faster in sds as well as normal page (and especially in urea-page).

since the mass scores are different, you can sequence the proteins to determine if there are any changes in the primary structure.

Edited by mdfenko, 24 May 2013 - 10:27 AM.

talent does what it can
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i do what i get paid to do




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