His-tagged protein, urea & antibody production

Koen De Smet k.desmet at nospam.ic.ac.uk
Tue Feb 3 07:24:05 EST 1998


Vladimir Svetlov wrote:
> 
> In article <34D5883B.CCF at nospam.ic.ac.uk>, k.desmet at nospam.ic.ac.uk wrote:
> 
>  > Glenda Lawrence wrote:
>  > >
>  > > Hi.
>  > >
>  > > Does anyone have ideas or experience re- the best
>  > > way to remove urea before injecting recombinant
>  > > protein into rabbits for antibody production?
>  > > How much urea can be tolerated?
>  > > The protein is a His-tagged protein (insoluble) purified
>  > > and eluted in 8M urea, 20mM Tris, 150mM NaCl and 200mM imidazole.
>  > >
>  > > Thanks,
>  > >
>  > > Glenda Lawrence
> >
> >
> > I don't know if this is the "best way", but we have succesfully produced
> > antisera by dialysing the urea away. In most cases, the recombinant
> > His-tagged protein preciptated, but that does not matter. YOu can still
> > make a suspension with incomplete Freunds adjuvant
> 
> Well, it does not have to precipitate. One of the simplest solutions is to
> do a step-wise dialysis: 6M->4M->2M->0.7M->0M urea. If you don't use higher
> than 0.5 mg/ml concentration most of the proteins are likely to stay
> solubilized. 

I tried that, but I had a protein that even precipitated in the 5M urea. But that doesn't 
matter for antiserum production. Finding the right conditions to refold into soluble, 
enzymatically active protein can be a pain. What works for one protein, doesn't work for the 
next one you purify.

Koen De Smet
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