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Ni affinity purification - not as simple as you may think


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4 replies to this topic

#1 milanoj

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Posted 18 January 2002 - 10:18 AM

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)


#2 Roger Moore

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Posted 06 March 2002 - 08:25 PM

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

#3 Muktikant

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Posted 16 April 2002 - 01:31 AM

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

#4 milanoj

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Posted 16 April 2002 - 06:34 AM

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


#5 Muktikant

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Posted 17 April 2002 - 12:35 AM

Milanoj,
Thanx for the suggestion.
Muktikant




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