Re: [ccp4bb] protein degradation?

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CCP4bb <-- 2007 <-- November 2007 <-- 04 November 2007

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Subject: Re: protein degradation?
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2007-11-04

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Subject: refmac5: FIND CONTACT WITH BRICKING failed
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-11-05

Subject: Re: protein degradation?
From: Juergen Bosch jbosch {- at -} U {- dot -} WASHINGTON {- dot -} EDU
Date: 2007-11-04

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

CCP4bb navigation

CCP4bb <-- 2007 <-- November 2007 <-- 04 November 2007

Previous message:
Subject: Re: protein degradation?
From: Jacob Keller j-keller2 {- at -} MD {- dot -} NORTHWESTERN {- dot -} EDU
Date: 2007-11-04

Next message:
Subject: refmac5: FIND CONTACT WITH BRICKING failed
From: Tim Gruene tg {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-11-05