Protein solubility after amm.sulphate precipitation

Chia Jin Ngee mcblab47 at leonis.nus.sg
Mon Apr 28 00:48:27 EST 1997


Koen A.L. De Smet (k.desmet at ic.ac.uk) wrote:
: 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.

Same problem here. Elution with imidazole resulted in precipitation of 
the protein. But elution with EDTA did not. Any explanation besides the 
protein not being folded properly?

In my case, I didn't freeze the eluted protein solution. I refrigerated 
it at 4 deg C and it precipitated overnight.

--

Jin Ngee, Chia		
(Genie, the OligoMan)
mcblab47 at leonis.nus.sg

Views and opinions expressed are solely mine and not of my employer's. 
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