Hi all,
I am co-expressing 2 proteins (cloned to pETDUET1) in E.coli to check their interaction. I did the induction and checked cell lysate on SDS-PAGE. Surprisingly I observed a induced protein band at the added molecular weight (nearly 95KDa) of both proteins instead of 2 separate bands (45kDa and 50KDa). Have anyone observed similar result?
Thank you
Is it possible that the genes for the two proteins were cloned into the vector in frame as a single open reading frame? Did you sequence the insert?
You should see separate bands.... you can't see protein co-complex until you cross-link protein by crosslinkers .... SDS disrupt any co-compound. so band at 95kd might not be your protein of interest. good luck
may be you have some problem during the reduction procedure. May be this proteins can interact and migrate as one band in non reducing buffer conditions, but if you used reducing conditions, then you have to see two bands.
I've come across a few protein complexes that survive SDS PAGE - it's not as rare as you might think. So it could be that you have a very tight complex. Are you boiling samples prior to the gel? You can add further reducing agents (if there might be disulphides), urea or guandinium chloride. If you want to see an example of a really stable complex expressed in E. coli have a look at this paper: http://onlinelibrary.wiley.com/doi/10.1111/mmi.13030/abstract
One other idea. We used to make urea SDS PAGE gels. You want to make a gel containing 8 M urea. You'll find out that you need essentially no water - so the gel will be Urea powder, tris buffer, SDS and acryl/bi-acryl (+APS/TEMED). It won't be a pretty gel but it does denature proteins beautifully. We used to do this for E. coli OM proteins which are very hard to denature and on normal SDS PAGE migrate as 2 or even 3 distinct species (different degrees of denaturation). I don't have a protocol to hand but you can probably find one online.
Is it possible that the genes for the two proteins were cloned into the vector in frame as a single open reading frame? Did you sequence the insert?
There is nothing extrange in these results and the possible explanations have been indicated in previous answer. To confirm comigration you can perform isoelectrofocusing (if pIs are differents), western (if antibodies available) or, more simple, mass spectrometry. In any case, and depending of the gel concentration and lenght, it is possible that the two proteins be not resolved. If you have both run them in two lanes of the gel and see the results.
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