[ccp4bb] protein degradation?

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: tricky molecular replacement
From: Jan Abendroth jan {- dot -} abendroth {- at -} GMAIL {- dot -} COM
Date: 2007-11-04

Next message:
Subject: Re: protein degradation?
From: Wataru Kagawa wkagawa {- at -} JOTA {- dot -} GSC {- dot -} RIKEN {- dot -} GO {- dot -} JP
Date: 2007-11-04

Subject: protein degradation?
From: Vijay Kumar vijay85 {- at -} GMAIL {- dot -} COM
Date: 2007-11-04

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

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: tricky molecular replacement
From: Jan Abendroth jan {- dot -} abendroth {- at -} GMAIL {- dot -} COM
Date: 2007-11-04

Next message:
Subject: Re: protein degradation?
From: Wataru Kagawa wkagawa {- at -} JOTA {- dot -} GSC {- dot -} RIKEN {- dot -} GO {- dot -} JP
Date: 2007-11-04