His- binding column

[Federici]

In article (Dans l'article) <4s6jim$7p1 at mark.ucdavis.edu>,
ez005679 at dale.ucdavis.edu ([user unknown]) wrote (écrivait) :

> I have a problem of purifying over expressed protein using His-binding 
> resins provided by Novagene company.  The problem is my protein does not 
> bind to the column, even after I tried urea denaturation.   The protein I 
> am trying to purify is a memebrane binding protein and was shown to be 
> present in the binding  and washing solutions after loading, but 
> not in the eluting solution.    Could anybody please give me some advice 
> on this column purification?   Thanks in advance
> 
> Yusen 

retention on affinity chromatography column is dependent upon the level of
affinity of your protein for the resin. A competition with other proteins
contained in the extract loaded, may hinder the binding. A purification of
the protein (before the loading on His-binding resin) will facilitate the
retention of your compound. You may try precipitation ( using ammonium
sulfate), followed by gel filtration or hydrophobic chromatography.