Some proteases are metal-dependent, and inhibitors for those aren't
Ni-column-compatible. "We" (meaning my students) found that it helps
to (1) work very quickly and (2) put EDTA into the fraction collector
tubes before eluting from the Ni column.
At 06:22 AM 11/4/2007, 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
---------------------------------------------------------------------------------------------------------------------------
Phoebe A. Rice
Assoc. Prof., Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
fax 773 702 0439
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
