glycerol to avoid protein precipitation?...

Achim Recktenwald achim at ibex.ca
Mon Aug 26 08:04:03 EST 1996


Bill Prichett wrote:
> 
> Arioch wrote:
> Arioch,
>     I read a long time ago in a protein purification and protein methods book that adding glycerol to a stock
> and freeze at -20 prevents a couple of things that help to maintain protein stability.  First, I prevents ice
> crystal formation.  Thus you do not have micro sheering stresses placed upon the protein.  You also do not have
> to warm the sample to take an aliquot. thus know freeze thaw cycles( which are probably an accumulation of both
> effects that I will mention).  We routinely add 1 volume of glycerol to all antibodies that we use (final conc.
> = 50% glycerol).  I have had antibody stocks that I go back into years later with out hesitating about it's
> quality.  Second, the book also mentioned that even in well buffered samples, as ice cystalizes, the
> microenvironment that the protein is in can change drastically.  pH shifts occur on a micro scale, salt
> concentrions as well as other additives, change (concentrate).  The book stated, I believe, that you could get
> a +/- 1-2 pH unit swing in a sample.  That could be as much as 4 units.  Some proteins can not handle so much.
>  I hope this helps.  As far as a reference I believe it was either "Protein Methods" by Bollag and Edelstein
> Pub-Wiley-Liss  ISBN 0-471-56871-6  or "Guide to Protein Purification" Ed Deutscher, Academic Press ISBN
> 0-12-213585-7 (This is also the same as methods in enzymology v182).  Good Luck,
> 


When a sample is frozen a kind of demixing occurs, the first ice is
almost ion-free. The buffer, salt and other components concentrate in
the still liquid part of the sample.Therefore, a quick freezing process
can often prevent damage of susceptible samples; shock-freezing them in
liquid notrogen is a good method.

Another important aspect is the choice of buffer used. Somebody already
mentioned this before in this thread. Tris is one of the worst possible
choices to store a frozen sample. The pH-shift of the buffer is
-0.028pH/K. That means if you freeze a sample having a pH of 8 at 25C in
a -70C freezer, the pH of the sample drops by 95*0.028K = 2.66pH-units.
I usually use phosphate-buffer to freeze samples; the pH of phosphate
buffer changes only 1/10th as strongly as Tris, i.e., - 0.0028pH/K. A
sample adjusted at 25C endures in phosphate-buffer only a pH-drop of
0.266pH-units.

Achim



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