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 >
