Let me correct myself, it is the imidazole buffer (not the nickel) and
boiling your protein in does the rest
I will try to find the exact reference for this phenomenon.
J.
Jeroen Mesters wrote:
Hi,
if I recall this correctly, it is the nickel that is in your sample
after elution and boiling your protein in SDS sample buffer does the
rest.....
So, could be the sample is fully okay!!!
J.
Tiago Botelho wrote:
Hi,
I also had a similar problem with one of my proteins... I had it cloned in
two different plasmids, one with Cter His-tag and the other in the Nter.
Whenever I purified it using IMAC purification I would get the double band
(that I confirmed by MS and were the same).
I got ride of this "double band" when I decided to simply avoid Ni-IMAC
purification and just use ion-exchange methods like Q/S Shepharose. It
seems that I had not degradation but simply some chemical alteration due
to the use of IMAC column.
Good luck and best regards,
Tiago.
---
Tiago Botelho
PhD Student
IBMB - CSIC
Institut de Biologia Molecular de Barcelona
carrer Jordi Girona 18-26,
08034 Barcelona
Phone: +34 93 4006100 ext. 269/332
Fax: +34 93 2045904
www.ibmb.csic.es
On Mon, November 5, 2007 4:01 am, Juergen Bosch wrote:
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
--
Jeroen Raymundus Mesters, Ph.D.
Institut fuer Biochemie, Universitaet zu Luebeck
Zentrum fuer Medizinische Struktur und Zellbiologie
Ratzeburger Allee 160, D-23538 Luebeck
Tel: +49-451-5004070, Fax: +49-451-5004068
E-mail: [EMAIL PROTECTED]
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.opticryst.org
--
If you can look into the seeds of time and say
which grain will grow and which will not - speak then to me (Macbeth)
--
