Ni-NTA problem - His-tag protein that never bound with Ni-NTA agarose (Mar/17/2005 )

Dear friends,
I am dealling with bacteria OMP. I had cloned the OMP gene into QiaExpress vector pQE30 and was N-terminal tagged with His. However, when I purify my protein, I can't get my protein in elution buffer, but they all present in washing buffer. What is happening?

Thank You

Regards
Yongyk

hi
i got this problem once and that was a problem of column equilibration...

well it was a long time ago. but i do remember problems occured in the begining with pH equilibration of the column. And it didn't retained the protein. But i quit the lab (it was a practice). I'm gona ask my boss to get more info.

cheers

Maybe you can change the his tag from one termini to the other. The tag might be covered into the protein and not accessible to the Ni-NTA beads.

If your protein form high MW aggregate, it will not bind to any column including Ni-NTA. To see whether your protein form aggregate, run your protein to a suitable gel filtration column and see whether it come out at void volume.

Dear friends,
Since I purify my protein in denature condition, will it still form agregate?
If it do form agregate, how to get rid of it?

Regards
Yong

Run suitable gel filtration in the presence of denaturant and see if your protein come out at void volume.

hy,

what is the composition of you buffer ? it is possible that your pH is not appropriate.. an acidic ph can prevent the binding. if your are using 10 mM imidazol to decrease ssome aspecific binding on the column, it can also prevent the binding of your protein to the resine. One possibility is to try to purify with 8M urea. you will be under denaturing conditions but your his tag will become accessible.

good luck

O2

i agree with Aude.
The pH of the buffer should be above 7.
I always run Ni column without imidazole in binding buffer because my protein did not bind column.
Also have you tried other metal ions for example Cu and Zn?