Ahh, yes, the "dancing crystals problem". The good news is alcohols are
really good cryoprotectants as well as excellent precipitants. Shame
their use has fallen out of favor over the years, but I guess as drop
sizes got smaller the evaporation problems got worse and worse.
In my humble opinion, this is just an extreme case of a problem we all
have already. Evaporation during harvesting is an insidious issue that
is hard to monitor. It's not just alcohol, but water that evaporates,
and some buffers are volatile too (like ammonium and acetate ions).
Volatile buffers mean the pH changes over time. All this can easily lead
to non-isomorphism between the first crystal you mount vs the last. An
excellent review is: https://dx.doi.org/10.1107%2FS1399004714012310
It has already been suggested that you surround your harvesting
environment with a wet towel (aka Kimwipe) soaked with the well
solution, and this is a good idea to try and keep the local humidity (or
alcohol-idity?) up. Another possibility is to make up 30-40 mL of your
well solution, put that into a 50 mL conical tube and use one of those
stone fish-tank aerators to bubble air or N2 gas through the appropriate
solution for generating just the right "atomosphere" that your crystals
are used to (see attached photo). You can then direct that gas through
an additional length of hose in the general direction of your well
before you crack it open. The point is to keep your crystals unaware of
the fact that they are about to be harvested for as long as possible.
In your case you have an excellent assay for when you have kept the
harvesting environment properly controlled: the crystals will stop dancing.
There are some devices on the market now, such as the "HC1" from Arinax,
or the "Watershed" from MiTeGen that are a much more sophisticated way
to do this, but check with the vendor before filling it with alcohol.
Some seals don't like non-aqueous liquids. I expect there may be a
safety concern about generating large amounts of alcohol vapor,
especially isopropanol. You don't want to breathe that in. Best to
keep away from open flames and work in a well-ventilated area, like the
hood. Ethanol is less toxic but on a per-capita basis more dangerous.
At the very least, don't drive yourself home afterwards.
-James Holton
MAD Scientist
On 8/14/2018 1:58 PM, Thomas Krey wrote:
Dear crystallization experts,
We have 3D protein crystals grown from a microseed matrix screening
vapor diffusion experiment in either
15% (v/v) Reagent alcohol
HEPES Na pH 7.5
0.2 M MgCl2
or in
27% Isopropanol
0.18 M MgCl2
90 mM HEPES Na pH 7.5
10% Glycerol
Upon opening the corresponding wells these crystals move quite a bit –
presumably due to the volatility of the alcohols. Does anyone have a
good suggestion to stabilize the swirling movements? Does anyone have
experience, whether these conditions alone can serve as
cryo-protectant (i.e., do we really have to fish, move into cryo
solution and fish again)?
Any suggestion or input would be highly welcome.
Thank you very much in advance.
Thomas
Prof. Dr. Thomas Krey
Hannover Medical School
Institute of Virology
Structural Virology Group
Carl-Neuberg-Str. 1
D-30625 Hannover
phone: +49 (0) 511 - 532 4308
email: krey.tho...@mh-hannover.de
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