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Problem in non-specific protein synthesis


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#1 BrettMIMR

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Posted 23 January 2005 - 10:06 PM

Hi all,
My problem lies in the synthesis of a non-specific protein that shows up every time I synthesise my HIS-tagged protein from my pET-21b plasmid in BL21 competent cells. What's happening every time I express and purify my protein a second protein which is smaller (5kDa) keeps showing up on a western when blotted with an anti-HIS Ab. The protein has been disregarded as a degradation product and because it contains a HIS tag from the westerns would consider it to be a second protein that is being made from my sequence. According to my sequence there are 2 ATG start codon sites further down the code which are in-frame from my initial ATG start codon site, which doesn't contain a Kozak sequence however has a ribosomal region near by. My start codon site is TAGATGG, now according to the consensus the site should be ACCATGG, would this be one of the reasons why sometimes I get my protein to read at this site and a second non-specific protein to read at another ATG site? Is the ribosomal region acting as a Kozak sequence? Is there a way of actually getting rid of this second protein by other means?
I would appreciate any response. :unsure:

Edited by BrettMIMR, 24 January 2005 - 04:58 PM.


#2 tuckern

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Posted 24 January 2005 - 01:48 AM

Run a gel, coomassie stain it, then cut out your mystery band. You can then use MALDI-tof analysis to establish what the protein is.




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