Hi,
Ii happened to me before.
The protein was a dimer and by denaturing the purified protein with
urea, and rerunning the Ni-NTA, I could separate the 2 species. So the
dimer was a mixture of cleaved and uncleaved protein.
If I recall correctly, we then observed degradation again after
refolding and assumed some kind of self proteolysis. We ended up
recloning the protein without the C-ter and had a homogeneous sample but
the crystals didn't diffract and the project stopped there so that's
about all I can say.
good luck
vincent
Vijay Kumar 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
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