HELP: dialysis of protein

[Jim Litts]

In article <1995Mar4.182321.1 at mscf.med.upenn.edu>,
pathology at mscf.med.upenn.edu wrote:

> >> NAME RAVI PRAKASH .D (sl16s at cc.usu.edu) wrote:
> >> : Dear Netters,
> >> : I have attempted to dialyse a recombinant protein (in 0.5M NaCl, 300mM
> >> : imidazole, 6M urea) into buffer with 150mM NaCl, 20mM tris, 
> 2.5mM CaCl2. Within
> >> : few minutes of dialysis there is a precipitate 
> formation in my dialysis bag.
> >> : Can anyone have any suggestions as to how I can rescue my protein from the
> >> : precipitate? Or any better way to dialyse the protein in future?
> >> : Thankyou in advance for any suggestions or comments.
> > 
> 
We have experienced similar problems with our proteins and have currently
adopted a general approach that has been successful in the two instances
we've tried so far.  That is to set up a series of dialysis solutions from
pH 6 to 9, with salt from 0 to 0.5 M, and plus or minus a detergent
(usually NP40, but octyl glucoside is generally better in dialysis) and
place 1 ml aliquots of our sample in each.  This approach has allowed us to
"map" a proteins "solubility space" quickly while only exposing a
relatively small amount to precipitation.  We have also taken samples that
precipitate and returned them to a non-precipitating condition to test for
reversibility of the precipitation.  The little 'slide-alyzer' dialysis
chambers from Pierce have been useful in this protocol, though I'm sure a
series of bags would be just as effective.  This approach takes some of the
guess work out of the experiment, and I have found it very difficult to
predict how a protein is going to behave based upon the behavior of other
proteins.  They're all so individual, you know...  ;-)

Good luck!

Jim