Another possibility, since you say MS looks identical and you are unable
to separate those two bands by other chromatografic means, is simple a
metal binding site in your protein. If the charge is changed in your
protein due to metal binding then the apparent molecular weight will
differ - that then could explain the identical results in MS (if I
understand what you say regarding the MS correctly, if the masses are
different, then forget about my sentences)
Try incubating your protein with e.g. EDTA and see if you get a single
band in your SDS PAGE.
Jürgen
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
--
Jürgen Bosch
University of Washington
Dept. of Biochemistry, K-426
1705 NE Pacific Street
Seattle, WA 98195
Box 357742
Phone: +1-206-616-4510
FAX: +1-206-685-7002
Web: http://faculty.washington.edu/jbosch
