HELP: dialysis of protein

pathology at mscf.med.upenn.edu pathology at mscf.med.upenn.edu
Sat Mar 4 18:23:21 EST 1995


>> 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.
> 

I suggest that you vary the ionic strength of your refolding buffer.  In other 
words, increase or reduce or eliminate the NaCl and/or CaCl2. Your protein may
remain soluble in  higher or lower ionic strength buffer.  A recombinant
protein that I am working with stays in solution with stepwise dialysis from
6M, 4M, 2M, 0M urea when in 40mM Tris, pH 8, as the buffering solvent; whereas
it precipitates when I put it through the same dialysis step with decrements of
urea concentration with PBS as the buffering solvent (100mM NaCl and 10mM
sodium phosphate, pH 7.5).  pH and the type of buffering chemicals can be
important, too.  The calcium may or may not interfere with solubility.  Don't
give up hope yet. 

Gregg Wells
Department of Pathology
University of Pennsylvania
Philadelphia, PA  19104-4283
email:  pathology at a1.mscf.upenn.edu




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