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: [log in to unmask]
Http://www.biochem.uni-luebeck.de
Http://www.iobcr.org
Http://www.opticryst.org
--
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--
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