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Re: [ccp4bb] protein degradation? |
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CCP4bb navigationCCP4bb <-- 2007 <-- November 2007 <-- 04 November 2007Subject: Re: protein degradation? From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL Date: 2007-11-04 > Are you expressing a eukaryotic protein? If so, you might want to > check for rare codons. There are a number of websites where you can > put in your coding sequence and check. I recently had this issue and > it turned out to be incomplete/stalled translation rather than > proteolysis as I had several rare codons in succession. You can > circumvent this by adding an antibiotic selectable plasmids encoding > the rare tRNAs. indeed thats often the case; there are commercial cells with plasmids encoding for rare codons, rosetta-2 work really well for us. A. > Good luck > Eric > > > On 11/4/07, Vijay Kumar >> Hi, >> >> I have been trying purify a N-ter his-tagged protein over- >> expressed in >> E.coli. After purification (Ni-NTA or Co-Talon), I find two bands >> in SDS >> PAGE which are very close each other (top band in the right MW and >> more >> intense than the lower band). Western blot (for his-tag) of the >> gel gave >> signal for both the bands. Mass spec results confirmed both >> protein bands >> are the same. So I think it could be C-ter degradation of my >> protein. Also >> the 2 bands exist after ion-exchange and sizing column. >> >> I use commercially available complete protease inhibitor tablets >> (increasing >> concentration has no effect) and sonication for lysis. I am >> wondering if >> people have encountered the same problem and got any suggestions? >> >> >> Thanks in advance. >> >> Regards, >> >> Vijay >> >> > > > -- > D. Eric Dollins, Ph.D. > C266 LSRC, Research Dr. > Duke University Medical Center > Durham, NC 27710 > (919) 681-1668, d.e.dollins@gmail.com CCP4bb navigationCCP4bb <-- 2007 <-- November 2007 <-- 04 November 2007 |
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