Ni-NTA purification

J. Arturo García-Horsman arturo.garcia-horsman at uku.fi
Mon May 7 03:03:42 EST 2001


John wrote:

> I have expressed my gene of interest (0.8kb) in a C-terminal His-tagged
> expression vector. I used 200mM Tris SO4 pH7.9 in resuspending the cell, and
> obtained the cell free extract by sonication.
>
> Then I would like to purify the protein by using Qiagen Ni-NTA spin Kit (Cat
> No. 31314). However I found that the eluate are the same as the portion
> saved from the washing step. That is all the bands came out instead of a
> single band.
>
> I have tried to resuspend the cell in the lysis buffer (as described in the
> protocol), however the result was the same.
>
> The expression of the protein of my interest has been confirmed by
> Activity-stained PAGE.
>
> So, would anyone give me some suggestions? Thank you very much.
>
> Johnny
>
> ---

Hello Johnny,

1) Is your protein still intact? Has the histidine tag remained at C-terminous?
You can check that, if you haven't, by western with anti-6Xhis antibody. It may
be that your protein is loosing the tag post-expression or during lysis

2) Are you loading your sample in low imidazol? something like 10-20 mM imidazol
in the loading buffer usually helps in avoiding unspecific binding.

3) How many times you washed your column? you can wash it more before elution to
eliminate all the unspecific binding. Is your protein present in all fractions?
(i.e. washes, eluate). Is it coming out at all from the column?

4) I guess that your avoiding all the components that could compete
for/inactivate the comunn.

5) . . .

Cheers,

Arturo.
_______________________________________________________________

Arturo Garcia-Horsman, Ph.D.
Research Specialist
Department of Pharmacology and Toxicology
University of Kuopio
Canthia, Harjulantie 1 A, 1. krs.
70210 Kuopio, Finland
Tel: +358-017-162422
Fax: +358-017-162424
E-mail: Arturo.Garcia-Horsman at uku.fi





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