IUBio

[Protein-analysis] pure protein (?) but two bands on SDS-PAGE

Rhiannon Evans evansrm8 at Cardiff.ac.uk
Fri Jul 28 03:11:58 EST 2006


Thanks, but I dont think there are any glycosylation pathways in E.coli and I
have also tried de-phosphorylation but it makes no difference. Are there any
other common post-translational modifications to consider? 

>>> "Amanda Gillespie" <gillespie.amanda.spamfilter at gmail.com> 27/07/06 6:14 PM
>>>
Some apparently pure proteins often run as doublets - this could represent
slightly different  glycosylation forms, or other post-translational
modifications, or may represent protein-protein interactions/dimerisation.
Its not uncommon.

On 7/25/06, Rhiannon Evans <evansrm8 at cardiff.ac.uk> wrote:
>
> Hi, I have cloned the gene for my protein into two pET vectors, one with
> an
> N-term His-Tag, one without. When I purify my recombinant protein from E.
> coli
> BL21(DE3)RIL Codon Plus cells via affinity chromatography (methotrexate or
> Nickle column) I get two bands close together at approximately the correct
> molculer weight for the target protein on SDS-PAGE. I have tried to
> denature the
> protein in 8M Urea, and reduced any disulphide bonds with mercaptoethanol
> and
> TCEP, but still get two bands. MALDI-TOF Mass Spec shows a peak at the
> correct
> molecular weight only. I am thinking there has been some sort of post
> translational modification?? Any suggestions would be appreciated. Cheers
> :-)
>
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