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.
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
