Purification of proteins in denaturants

Norma Greenfield greenfie at umdnj.edu
Wed Sep 18 15:36:53 EST 1996


zaphod at mcimail.com (Blake Middleton) writes:

>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
Guanidine HCl is extremely ionic, and therefore proteins will not stick to
ion exchange resins in this denaturant.  However, there is no reason why the
protein shouldn't  bind in the presence of urea.  In our laboratory 
we run both DEAE and CM columns in the presence of urea.  
	You could also try size exclusion chromatography using HPLC or FPLC.




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