7 replies to this topic

[yongyk]

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

[fred_33]

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

[yongyk]

fred_33, on Mar 17 2005, 02:47 AM, said:

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

<{POST_SNAPBACK}>

Hi,
Could you be more specific?

Thank

Yongyk

[fred_33]

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

[GoGoalGirl]

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.

[leekaming]

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.

[yongyk]

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

[leekaming]

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