On Nov 4, 2007, at 14:23, Eric Dollins wrote:
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 <[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]
