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. Good luck Eric
On 11/4/07, Vijay Kumar <[EMAIL PROTECTED]> wrote: > 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, [EMAIL PROTECTED]
