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 >> >>
