Purification of proteins in denaturants

Achim Recktenwald achim at ibex.ca
Tue Sep 17 12:22:49 EST 1996


Blake Middleton wrote:
> 
> 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 should have no problems doing ion-exchange chromatography with urea;
did it myself for months on a FPLC. 

But you should be careful with your pumps. Make sure there is no leakage
at the seals or you have too much crystallization of urea. I did this
kind of chroatography for about 6 months. The only damamge on the FPLC
was a set of exchange-seals. And you should renew your pre-filter before
you flood the system with 6M urea, otherwise you dissolve all the
accumulated junk since the last change.

As gels I used Mono-Q, Mono-S, and later Q-Sepharose FF - no problems.


Guanidine you could use only in a gel-filtration, but you should keep in
mind that you would need a lot of the stuff just to equlilbrate your
column; this might be a bit expensive.


Gooid luck,

Achim



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