4 replies to this topic

[milanoj]

Has anyone had problems with Ni affinity purification?

(Edited by milanoj at 4:20 am on Jan. 18, 2001)

(Edited by milanoj at 4:21 am on Jan. 18, 2001)

[Roger Moore]

I have an enzyme overexpressed in the pET28 vector. The crude protein extract after sonication shows activity. then I put it through the Ni-NTA Qiagen column (by FPLC) and get one beautiful band by SDS-Page at the right molecular weight. Unfortunately, it has no activity after being exposed to the Ni resin.
Roger

[Muktikant]

I will be tryng this method. Do you think Antigenic determinants will remain conserved after exposure to Ni column.

[milanoj]

007,
I have had this problem also. Reactions conditions are all important. Check for active sulfhydrils. I am not an expert at this stuff but a good primer is R.K. Scopes - Protein purification, principles and practice (Springer Press)

Muktikant,
It all depends on folding post-purification. You can purify under gentle non-denaturing conditions that should maintain the proteins secondary structure. If you are using inclusion bodies you'll need refolding conditions. See the above reference

[Muktikant]

Milanoj,
Thanx for the suggestion.
Muktikant