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Ni-NTA-Columns

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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?
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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.
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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)
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Re: Ni-NTA-Columns

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

thank you. I think I will try the other columns :wink:
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Re: Ni-NTA-Columns

Postby chance » Thu Oct 15, 2015 9:13 am

1. Imidazole concentration is too high. When eluting with imidazole, each gradient must elute completely;
2. The cleaning of sample is not sufficient, unable to remove unbound contaminants. You can extend the cleaning time and increase the imidazole concentration to improve the cleaning effect;
3. Increasing the concentration of sodium chloride to reduce the non-specific binding;
4. The target protein production is mixed with non-tagged proteins.
You can use tris buffer instead of phosphate buffer to reduce non-tagged protein binding.
http://www.biologicscorp.com/
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