----- Original Message -----
From: "Juergen Bosch" <[log in to unmask]>
To: <[log in to unmask]>
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 : [log in to unmask]
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