Hi Thomas
One problem with conventional approaches is that you tend to get high
mosaicity when harvesting from drops with isopropanol.
A very good method, invented by my old friend Lesley Haire, that has always
worked (so far!) is to use microbatch-under-oil with a plate called the
Vapor Batch plate.
- Set up the drops with everything in them except for the isopropanol.
- Cover the drops with 50:50 paraffin and silicone oil ("Al's Oil").
- Put a few mL of 27% isopropanol in the "moat" around the outside of
the plate.
The isopropanol will saturate the oil and then diffuse into the drops. You
can take your time and harvest the crystals through the oil. The oil
protects the crystals, preventing evaporation of isopropanol.
Let me know if you (or anyone) would like some sample plates to try this
out.
See example and ref. below.
Best wishes, Patrick
___________________________
Example: https://www.douglas.co.uk/winner1.htm
*G. B. Mortuza et al.. High-resolution structure of a retroviral capsid
hexameric amino-terminal domain. Nature 431 (2004), pp 481-485. (This
paper describes the use of the vapor batch plate with isopropanol.)*
Sent from mobile
Patrick Shaw Stewart
+44 7901 548 201
On Wed, 15 Aug 2018, 08:41 Yvonne Thielmann, <yvonne.thielm...@biophys.mpg.
de> wrote:
> Dear Thomas,
>
> maybe you can try to overlay your drop with LV CryoOil from Jena
> Bioscience. Then the evaporation of the solvent is slowed down and the
> crystals are directly cryoprotected when you move the crystals through
> the oil. We had quite good results when we cryoprotected crystals in
> this oil.
>
> Best wishes,
> Yvonne
>
>
> --
> Dr. Yvonne Thielmann
> Max Planck Institute of Biophysics
> Molecular Membrane Biology
> Max-von-Laue-Strasse 3
> 60438 Frankfurt / Main
> Germany
>
> Office +49 69 6303 1056
> Lab +49 69 6303 1074
> Fax +49 69 6303 1002
>
> Am 15.08.2018 um 08:05 schrieb Kajander, Tommi A:
> > Yes sorry, i meant paratone-N also.
> >
> > Tommi
> >
> > Kohteesta: ferrer
> > Lähetetty: keskiviikko 15. elokuuta klo 0.41
> > Aihe: Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant
> > Vastaanottaja: ccp4bb@jiscmail.ac.uk
> >
> >
> > Dear Thomas,
> >
> > Alternatively you can try shooting on crystals in the drop, in situ. So
> > fishing, no cryo. But potentially high radiation damage. Can be
> > considered if you have enough crystals, and if your crystallization
> > plate makes it possible.
> >
> > Regards
> >
> > JL
> >
> > On 14/08/2018 20:58, 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 <mailto:krey.tho...@mh-hannover.de>
> >
> >
> > To unsubscribe from the CCP4BB list, click the following link:
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> >
> > -- -------------------------------- Jean-Luc Ferrer Institut de
> Biologie
> > Structurale 71 Avenue des Martyrs CS 10090 38044 Grenoble Cedex 9 -
> > FRANCE Ph.: +33 (0)4 57 42 85 22 Cell: +33 (0)6 89 45 13 57 email:
> > jean-luc.fer...@ibs.fr <mailto:jean-luc.fer...@ibs.fr>
> > --------------------------------
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