Florian Schmitzberger wrote:
Dear Toby,
I don't think there is a basic problem using glycerol in
crystallization. Glycerol will affect the vapour pressure (if it is not
present in the well/precipitant solution) and 10 % glycerol is ~ 1.3
molar concentration. During equilibration the drops may increase in
volume, decreasing the protein concentration. Thus, when using glycerol
I think it is generally beneficial to start with a high protein
concentration. Perhaps, you can concentrate your protein sample further.
It sounds like you are encouraging the OP to crystallize by reverse
vapor diffusion. Unless there is reason to believe that diluting the
droplets will push the protein toward saturation (salting-in region?),
it would seem better to skip the reservoir, seal the wells and crystallize
by batch method. Even more sensible, (as you parenthetically hint) add
the same concentration glycerol to the reservoir solution to compensate
and crystallize by normal forward vapor diffusion.
And if you are lucky you can use the glycerol-containing reservoir
solution as artificial mother liquor if you need it for mounting
crystals or mixing cryoprotectant solutions.
Still, it makes sense to use higher protein or PEG to counteract the
tendency of glycerol to increase solubility/decrease nucleation.
I have on several occasions observed immediate precipitation upon mixing
protein solution (containing glycerol) and precipitant solution; drops
then cleared up after a short period of time (and crystals eventually
formed). In this case, the crystallization experiment starts in the
supersaturated zone, and "moves" towards an undersaturated
concentration, traversing the (metastable) zone where nucleation and
crystallization can happen (rather than the other way around, which
seems the more traditional approach with crystallization by vapour
diffusion).
Enrico Stura published a recent article, describing an effect of
glycerol on crystallization. Vera,L., Czarny, B., Georgiadis, D., Dive,
V., Stura, E.A. (2011) Practical Use of Glycerol in Protein
Crystallization. Cryst. Growth & Des. 11 :2755–2762. "
You could replace glycerol with ethylenglycol or a small molecular
weight PEG (e.g. 400), which may also have a stabilizing effect on your
complex.
Regards,
Florian
On Apr 3, 2012, at 7:49 AM, Toby Longwood wrote:
Dear all,
My question is related to a sample preparation.
I’m working with a complex that can be stabilized with glycerol (at
least 10%) during purification. The use of detergents does not help.
After purification, the sample is homogeneous (EM) and can be
concentrated (3-4mg.mL-1) . I already set up many drops, changing
several conditions (pH, salt...) but nothing conclusive appeared.
I know that crystallogenesis in presence of glycerol works (Sousa,
Acta Cryst (1995), ...) however, because of the aspect of the drops
(precipitates that seem close to the nucleation phase), I suspect that
the glycerol can be one of the limiting factors of the protocol.
Has anybody else been already confronted to the same problem? Does
someone know if there is an alternate additive to glycerol?
Thanks in advance for suggestions/help
With best wishes
Toby
