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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: homology modeling----good bond lengths, bad angles
From: anagha {- dot -} gupta {- at -} GMAIL {- dot -} COM
Date: 2007-02-28 		Next message:
Subject: Re: Cannot running NTA to purify the protein having His-tag?
From: aberndt {- at -} MRC-LMB {- dot -} CAM {- dot -} AC {- dot -} UK
Date: 2007-02-28


Subject: Re: Cannot running NTA to purify the protein having His-tag?
From: pinotsis {- at -} EMBL-HAMBURG {- dot -} DE
Date: 2007-02-28
----- Original Message -----
From: "Juergen Bosch"
To:
Sent: Wednesday, 28 February, 2007 8:18 PM
Subject: Re: [ccp4bb] Cannot running NTA to purify the protein having
His-tag?


Ngo Duc Tri wrote:

> 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
>
You His tag is most likely inaccessible, can you easily change the tag
from e.g the N-terminus to the C-terminus ? Or if you have a structural
homolog you could add the His tag into a loop, which is exposed.

Alternatively you can purify your protein under denaturing conditions
using 8 M urea and refold it if you dare :-)

Juergen


Or try a partial unfold of your protein by including 1-3 M Urea in your
buffer A

Nikos


*************************************
Nikos Pinotsis, PhD
EMBL-Hamburg, c/o DESY
Notkestr. 85, Geb. 25A
22603 Hamburg, Germany

Phone : +49 40 89902144
Fax : +49 40 89902149
e-mail : pinotsis@embl-hamburg.de
*************************************



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