Re: [ccp4bb]: is glycerol more bad than good

[Phraenquex VD <loretta@scripps.edu>]

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A better solution, in my opinion, is not to add glycerol at all:  instead,
concentrate it to something obscene, like 20 mg/ml, and freeze it in
aliquots.  Usually it acts as its own cryoprotectant at those
concentrations. 

This has the advantages that you have the identical protein available
months later, and you don't have to do odious glycerol extraction with
small volumes (which will also make aliquots differ). 

Of course, I know there are exceptions, and not all proteins concentrate
that high.  (Sux.)  But it's what I try first.  

phx.






On Mon, 7 May 2001, Bernhard Rupp wrote:

> Date: Mon, 7 May 2001 09:28:19 -0700
> From: Bernhard Rupp <br@llnl.gov>
> To: Ccp4 <ccp4bb@dl.ac.uk>, Bob Cudney <Bob@hrmail.com>,
>     CrystalNews <tech@hrmail.com>, Kim Henrick <henrick@ebi.ac.uk>
> Subject: [ccp4bb]: is glycerol more bad than good?
> 
> ***  For details on how to be removed from this list visit the  ***
> ***    CCP4 home page http://www.dl.ac.uk/CCP/CCP4/main.html    ***
> 
> Dear All :
> 
> I have a not crystallographic computing related but still interesting
> question. As often in protein crystallization, firm and validated
> information
> is rare and thus in this case I am happy to solicit also opinion and
> anecdotal evidence:
> 
> Glycerol is used to protect proteins while being stored frozen. This
> is a particular issue for any high throughput operations, where the
> protein cannot be processed immediately and needs to be stored in
> aliquots until machine time becomes available.
> 
> Now, the question is, how high a price will you have to pay later in
> crystallization success rate if you do not dialyze the glycerol out? I.e,
> what
> is the overall statistical chance that it is harmful vs. not?
> In particular, has reduced diffraction quality (vs.non-gycerol) been
> observed?
> I clearly understand that some proteins do crystallize fine with
> glycerol as additive, and we have it also in CRYSTOOL, but as a
> principal component in the protein stock,
> at lets say 10%, what's the effect? Does anybody have hard numbers
> (or some statistics) on that or at least more than single case evidence for
> the one or the other?
> 
> Electronic web research in Medline and inspec did not provide a lead.
> Manual search in J. Crystal Growth (1889-90) where we hoped to find an
> article presented at the first ICCBM conference in 88 was negative.
> 
> Please let us know if you can help with any information, references or
> leads.
> 
> I'll post the summary.
> 
> Thx, BR
> -----------------------------------------------------------------
> Dr. Bernhard Rupp
> Macromolecular Crystallography and Structural Genomics
> LLNL-BBRP L448                               Phone (925) 423-3273
> University of California                     Phax  (925) 424-3130
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