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Re: [ccp4bb] protein degradation? |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: protein degradation? From: mesters mesters {- at -} BIOCHEM {- dot -} UNI-LUEBECK {- dot -} DE Date: 2007-11-05 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: mesters@biochem.uni-luebeck.de 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) -- CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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