Protein solubility after amm.sulphate precipitation

Koen A.L. De Smet k.desmet at ic.ac.uk
Fri Apr 25 06:22:55 EST 1997


Malini Madiraju wrote:
> 
> Dear netters,
> 
> I have a unique problem. My protein doesn't to go into solution after
> ammonium sulphate precipiation even though it was soluble before
> precipitation.
> 
> I do the following before amm.sulphate precipitation: Elute the protein
> from nickel affinity column, dialyse it into a buffer (8mM imidazole,
> 500mM Nacl, 10% glycerol, Tris-Hcl, 20mM, pH 7.5). Protein is soluble at
> this point. Then I do amm. sulphate precipitation. The ppt is now
> insoluble in the same buffer. I do ammonium sulpahte precipitation to
> concentrate the protein.
> 
> I welcome any comments, suggestions or references that could help me
> solve this problem. Thank you.
> 
> Malini R. Madiraju


I am not sure if this is related, but you may like to know this anyway.  
I once had a protein expressed in the pQE system and it was soluble. 
After purification,  I froze the fractions in the buffer they were in. 
(I cannot get hold of the details immediatly, because this was years 
ago, but it probably was the buffer recommended by QIAGEN, for native 
purification using imidazole elution).
When I took it out of the freezer and thawed it, most of it was 
precipitated. I could get it soluble again by adding NaCl to 1M.
I assumed that it precipitated because it had not folded properly, but 
was still soluble enough not to form inclusion bodies.

Koen
-- 

_______________________________________________________________

        Dr Koen A.L. De Smet
        Research Fellow
        Department of Medical Microbiology
        Imperial College Medical School at St Mary's
        Norfolk Place
        London W2 1PG
        Great Britain
                                     
        Tel: (+44)-(0)171-594 3946  
        Fax: (+44)-(0)171-262-6299   
        Email: k.desmet at sm.ic.ac.uk
	       http://www.sm.ic.ac.uk/medmicro/home.htm
_______________________________________________________________

"If we knew what it was we were doing,
 it would not be called research, would it?".   --   A.Einstein
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