Ni-NTA-Columns

[piefke]

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?

[blcr11]

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.

[BioLad]

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)

[piefke]

thank you. I think I will try the other columns