refolding denatured proteins

Stephan Witte stephan.witte at
Sat May 20 04:30:16 EST 1995

In article <3pi1tn$l37 at>, "Aron B. Jaffe"
<jaffe at> 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

I think the use of detergent will be a good idea. I did the purification
(in GuHCl) and the successfull renaturation of a very hydrophobic protein
expressed in e. coli as includiobodies.
I did all the purification in denaturing conditions, then i dialysed the
protein against 2M Urea/40 mM Tris pH 7.2, to allow partial refolding.
Then i added n-octylglucosid and removed the remaining urea by a simple
gel-filtration (Pharmacia PD10).
N-octylglucosid (i.e. from Sigma) is a very mild detergent with a very high
CMC. This allows the exchange or removal of detergent by simple dialysis. I
had no problems using the protein-detergent solution for MALDI,
immunization of rabbits and in cell-cultures.

good luck, Stephsn


Stephan Witte
Inst. of Immunology
University of Constance
stephan.witte at

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