Purification of proteins in denaturants

Federici federici at icgm.cochin.inserm.fr
Fri Sep 27 04:06:23 EST 1996

In article (Dans l'article) <zaphod-1609961705220001 at>,
zaphod at mcimail.com (Blake Middleton) wrote (écrivait) :

> Does any one have any good suggestions for purifying proteins in
> denaturants (e.g.,6M urea, 4M guanidine)?  I have a his-tagged protein
> which I've been able purify to a great extent by Ni affinity, but I'd like
> to have another chromatographic step.  The protein is only soluble in 6M
> urea or 4M guanidine.
> Sizing columns work poorly in these buffers and we have not found an
> ion-exhange resin to which the protein will bind in these buffers, either.
> Thank you.
> -- 
> Blake Middleton        Internet:  zaphod at mcimail.com
> MCI ID # 465-2423        Bitnet:  zaphod%mcimail.com at cunyvm.cuny.edu
>   "Ah, this is obviously some strange usage of the word `safe' that I
>   wasn't previously aware of."                    --Arthur Dent

You can test the solubility of your protein in a 10mM sodium phosphate
buffer (pH 6,8), 0,1% SDS, 1 mM DTT,  or in the same buffer added with an
increasing concentration of urea until you find the good conditions. Then
load on an hydroxyapatite column equilibrated with the buffer you have
found, and elute with a gradient of  sodium phosphate (10 mM - 500mM).
Usually, the lower the urea concentration is, the better will be the
retention; Absence of urea would be the best conditions...

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