Re: [ccp4bb]: PEG/isopropanol crystals

Title: Re: [ccp4bb]: PEG/isopropanol crystals

Hello ccp4-ers,
Sorry for yet another non-ccp4 related question.

We have beautiful crystals grown from 9% PEG4000 and 6% isopropanol.
I anticipate that they may be difficult to freeze in the loop due to the evaporation of isopropanol.

Does anyone have any suggestions for cryocooling these crystals.
I've thought to try the various oils. But perhaps we need to substitute the isopropanol
with another organic solvent by soaking.

Has anyone had any success cryocooling isopropanol grown crystals?

Thank you in advance, Gloria

I'm going to assume you are doing a hanging-drop experiment. Try transferring the crystallization setup to a 4 degree cold room--do this approximately 40 minutes to one hour before you transfer the crystals to a cryosolution. If you leave the plate in the cold room for too long, your crystals may dissolve or you may get more nucleation. Once you open the well, the evaporation will start, and as a result the crystals will dance furiously around in the well, making it difficult to secure a single crystal in a loop. Though this is frustrating, the effect is actually not as bad at 4 degrees--they move slower at the colder temperature (unfortunately, so will you). If you use a viscous cryosolvent (e.g., ethylene glycol, glycerol), the viscosity will increase at 4 degrees, so once you transfer the crystal to this solvent the movement will slow down dramatically. I've found it easier to use a high-viscosity, low vapor pressure cryosolvent than to use higher concentrations of isopropanol alone as the cryoprotectant. You will still need to work quickly, as the evaporation of the isopropanol will affect your cryoprotectant solution, and you may still have problems with ice crystal formation.

Diana

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Diana R. Tomchick
Assistant Professor
University of Texas Southwestern Medical Center
Department of Biochemistry

5323 Harry Hines Blvd.

Tel: +1 214 648 9760
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Dallas, TX 75390-9038, U.S.A. Email: Diana.Tomchick@UTSouthwestern.edu