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Ni-NTA problem


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7 replies to this topic

#1 yongyk

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Posted 17 March 2005 - 12:47 AM

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

#2 fred_33

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Posted 17 March 2005 - 01:47 AM

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

#3 yongyk

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Posted 21 March 2005 - 09:54 PM

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

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Hi,
Could you be more specific?

Thank

Yongyk

#4 fred_33

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Posted 22 March 2005 - 12:59 AM

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

#5 GoGoalGirl

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Posted 25 March 2005 - 11:13 AM

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.

#6 leekaming

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Posted 26 March 2005 - 08:42 AM

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.

#7 yongyk

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Posted 27 March 2005 - 06:57 PM

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

#8 leekaming

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Posted 28 March 2005 - 02:54 AM

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




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