protein precipitates:HELP !

Clemens Suter-Crazzolara un691cs at genius.embnet.dkfz-heidelberg.de
Fri Oct 7 03:27:37 EST 1994


I wrote:
> >However, if I then concentrate the protein (I put the dialysis
  > >tube on PEG15000) the protein precipitates like crazy.

   to which Klaus Knoblauch answered:
 > 
> I used PEG always for one purpose only and that is to precipitate 
> Proteins (i.e. ribosomes, phage-particles or large proteins).

my dialysis tubig has a COMW of 3500, so my impression is that the PEG
can't enter the tube. AM i right ?
 

> 
> for concentrating proteins after dialzsis, dry Sephadex G100 always
> worked fine for me, just put the dialysis tubing into the powder, and keep
> at 4oC, remove the Sephadex-coating from time to time (it forms a 
> chewing-gum-like coat onto the dialysis tubing. I've even one time dried 
> up a sample completely (forgot the sample ON) and was able to recover
> it by just rinsing the dialysis tube with suitable buffer.

 sounds good, I will try this for the next preparation. But could it be
 that your protein simply doesn't precipitate so fast ? If I remove the
 Urea from the mix by dialysis, I already observe a precipitate, this
 effect can be supressed by Tween-20 however.
 > 
> >I need this protein at very high concentrations, like 10 
> >mg/ml (quite a lot).C
> >What can I do ? most literature says that top circumvent
> >precipitation, the best is to keep the protein in dilute form.
> >can a precipitated protein regain its function if I would 
> >inject it in an animal, or add it to a cellculture ???
> 
> to gain what? for rabbit injection, in order to raise antibodies, I've 
> had samples in 8M Urea, when using Freud's Adjuvent, there's no problem
> as far as the urea is concerned.
> 
> 		hope this helps,
> 			Klaus

Ah, but I need the active protein, because we want to study its effect
in vivo, that is in the animal. Thus the high concentration of at least
10mg/ml is essential. Can a precipitated protein regain function once in   
the bloodstream, or how can I circumvent precipitation at the start ?

thanks again, clemens




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