Ni-NTA purification

John kbsb7s04 at hkem.com
Mon May 7 02:02:47 EST 2001


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


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