refolding denatured proteins

Bruce Appel appel at uoneuro.uoregon.edu
Fri May 19 15:27:12 EST 1995


In article <3pi1tn$l37 at netnews.upenn.edu>, "Aron B. Jaffe"
<jaffe at a1.mscf.upenn.edu> wrote:

> I am purifying a his tagged protein under denaturing conditions
> (6M GuHCl) and after dialyzing the Gu away my protein is 
> precipitating.  I am trying to remove the Gu slowly (dialyzing 
> against reduced amounts of Gu, i.e. 3M, 1.5M, .75M, etc, before
> dialyzing against PBS without Gu), but this doesn't seem to help.
> I have also heard that detergent should help, but I tried a buffer
> with NP-40 in it, and it still precipitated.  
> 
> Does anyone have any suggestions?
> 
> Also, if I use detergent to keep the protein in a soluble form, 
> can I use it in tissue culture experiments?
> 
> thanks
> 
> aron


Aron,

I once experienced a similar problem. In addition to what you described, I
tried various methods to renature the protein on fractionation columns. In
the end, the only thing that worked for me was to dilute the denatured
protein with buffer having no denaturant. By adding the protein to the
non-denaturing buffer, I reduced the concentration of denaturant to a
point that allowed refolding and, presumably, minimized interactions
between proteins that were causing them to precipitate. That leaves the
problem of concentrating the refolded protein. I found it sufficient to
use Centricon spin filters. I was then able to remove remaining GuHCl by
dialysis.

Bruce

-- 
Bruce Appel
University of Oregon
appel at uoneuro.uoregon.edu



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