Login

Join for Free!
114100 members


Ni-NTA-Columns

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

Ni-NTA-Columns

Postby piefke » Wed Feb 13, 2008 7:38 am

Hi!
Did anybody gain experience with protein purification with Ni-columns?
I am purifying a soluble protein of 50 kDa under native conditions. I use the columns of qiagen. Now I have the problem that my protein elutes with contaminants. I increased the stringency of the binding and washing buffer and eluted the last time with 200mM Imidazol in the first step and with 350 mM Imidazol in the second elution step. But there are still contaminants. What is the maximum Imidazol concentration in the elution buffer or are there any other tricks to get rid of these contaminants?
piefke
Garter
Garter
 
Posts: 24
Joined: Tue Sep 18, 2007 8:41 am

Postby blcr11 » Thu Feb 14, 2008 12:09 pm

If you're using an fplc instrument, you're better off using a gradient of imidazole for elution. Then your stuff will elute at whatever minimal concentration of imidazole that suits it. You can also try using some other chelation matrix. USB, for one, sells a silica-based Ni resin they claim gives lower non-specific binding compared to either IMAC or agarose-type resins.
blcr11
Viper
Viper
 
Posts: 672
Joined: Fri Mar 30, 2007 4:23 am

Re: Ni-NTA-Columns

Postby BioLad » Thu Feb 14, 2008 4:49 pm

I'm with Piefke here about gradients. I used USB's PrepEase kits and they work great (http://www.usbweb.com/category.asp?spec ... 5&id=78794)
User avatar
BioLad
Garter
Garter
 
Posts: 14
Joined: Wed Nov 21, 2007 6:58 pm


Re: Ni-NTA-Columns

Postby piefke » Thu Feb 21, 2008 8:09 am

thank you. I think I will try the other columns :wink:
piefke
Garter
Garter
 
Posts: 24
Joined: Tue Sep 18, 2007 8:41 am


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 1 guest

cron