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Re: [ccp4bb] Cannot running NTA to purify the protein having His-tag?
 

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CCP4bb <-- 2007 <-- February 2007 <-- 28 February 2007
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Subject: Re: Cannot running NTA to purify the protein having His-tag?
From: sabrina {- dot -} biarrotte-sorin {- at -} MAIL {- dot -} MCGILL {- dot -} CA
Date: 2007-02-28
Hi,

I would try to include up to 1 to 2 M NaCl in your lysis buffer and
during purification .... you can then decrease your salt concentration
in your elution buffer ...
Good Luck
Sabrina Biarrotte-Sorin


Quoting Ngo Duc Tri :

> Dear CCP4 users,
>
> I'm purifying a kind of protease having His-tag. The protein is expressed in
> insect cells and broken by sonication.
> I used NTA resin to purify this protein.
> Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl. Buffer B is 50mM
> phosphate buffer pH 7.5, 300 mM NaCl and 300 mM Imidazole.
> However, all proteins cannot bind to NTA resin. My protein is eluted in
> Flow-through. I also check the NTA resin with the control His-tag. The
> western blot also shows that my protein has His-tag.
>
> Do you have any ideas about my problem? I'm really appreciate all of your
> advices how to solve this. Thank you very much!
>
> My best regards,
> TriNgo
> Sungkyunkwan University
>



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