Re: [ccp4bb]: PEG/isopropanol crystals

[Axel Innis <axel@ncbs.res.in>]

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Dear Gloria,

We had some long, thin, needle-like crystals growing in 30-35% isopropanol
and 0.6-0.7 M ammonium acetate (16 degrees), which made evaporation a real
problem. In order to minimize damage to the crystals, we used the
following protocol:

The contents of the hanging drop were transferred into a shallow well
containing 200 microliters of the mother liquor, either by using a large
loop or simply by washing the drop off the coverslip with mother liquor
(perhaps a bit violent, but it worked for us). After this, the best
crystals were selected and transferred to a fresh 200 microliter drop of
mother liquor using a loop. In order to minimize damage due to isopropanol
evaporation, we kept replacing half of the well solution with fresh,
temperature-equilibrated mother liquor every five minutes.

Cryoprotection was then achieved through the stepwise addition of
increasing glycerol concentrations to the well. After each addition, the
well was sealed with a coverslip and its contents were left to equilibrate
for 5 minutes. Once the correct concentration of cryoprotectant was
reached, crystals were frozen immediately in liquid nitrogen.

Crystals which were treated in this way diffracted to 1.5 angstroms and
had a mosaicity of 0.2-0.3 as opposed to 1.0-1.5 for those for which the
"5 minute" rule was not applied.

I hope that this helps in some way.

Regards,

Axel



On Mon, 27 Jan 2003 gborgstahl@unmc.edu wrote:

> 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
> 
> ******************************************************************************
> Gloria Borgstahl 
> Eppley Institute for Cancer Research
> 987696 Nebraska Medical Center 
> 10732A Lied Transplant Center 
> Omaha, NE 68198-7696
> 
> Office (402) 559-8578
> FAX (402) 559-8577
> 
> Associate Professor 
> Interests:  Cancer, Biochemistry, DNA Metabolism, 
> Protein Crystallography, Super-fine phi slicing, Synchrotron 
> Radiation, Crystal Mosaicity, and X-ray Topography
> ******************************************************************************

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_________________________________________

 AXEL INNIS

 National Centre for Biological Sciences
 Tata Institute of Fundamental Research
 UAS-GKVK Campus
 Bangalore 560 065
 India

 Phone +80-3636421/8  Extn. 4241 

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