precipitation problems

Michael G. Kurilla mgk2r at UVA.PCMAIL.VIRGINIA.EDU
Sat Nov 4 13:13:10 EST 1995


On Nov 4, 11:33am, Lim Pohyam wrote:
> Subject: precipitation problems
> My bacterial recombinant proteins precipitates upon long storage at -20C. 
> Upon thawing from freezer, I detected strands of precipitates. There is 
> difficulty to re-suspend these strands. As a result, when I set up my 
> microtiter plates, the activity is decreased. I sometimes encounter as much
> as 15-20% drop in protein concentration. This is creating havoc in my 
> assays. I would like to receive feedback on how to remove this problem.   
> what can I do? I keep losing my protein everytime I thawed them from the  
> freezer! 
> 
> 
> -- End of excerpt from Lim Pohyam <pohyam at technet.sg>

Freezing at -20C does not in reality always freeze. Anything dissolved in
water will depress its freezing point. What can happen with proteins,
particularly concentrated solutions is that as the solution freezes, the
protein will tend to migrate to the remaining fluid regions and gradually
become superconcentrated. Salts and buffers will also concentrate and the
salt conditions or the pH can be drastically altered. 

In fact, at -20C, the proteins are not really frozen, rather there is a thin
shell of fluid with concentrated salts and buffers. This can promote
denturation or degradation of the protein over long term storage.

Several options would include
     1)   freezing and storage at -70C
     2)   quick freezing in LN2 followed by -20C storage
     3)   adding glycerol to prevent freezing completely and storing at -20C

Mike Kurilla



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