Re: [ccp4bb] Freezing under oil

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Freezing under oil
From: Jim Pflugrath Jim {- dot -} Pflugrath {- at -} RIGAKU {- dot -} COM
Date: 2010-02-04

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Subject: EMBO Course on Macromolecular Complexes: Grenoble, 31 May- 5 June 2010
From: Carlo Petosa carlo {- dot -} petosa {- at -} ESRF {- dot -} FR
Date: 2010-02-04

Subject: Re: Freezing under oil
From: Roger Rowlett rrowlett {- at -} COLGATE {- dot -} EDU
Date: 2010-02-04

http-equiv="Content-Type">

Sometimes glucose works when glycerol or ethylene
glycol does not. We have also had very good success with a gradual,
in-the-drop cryo method: see
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Mounting+Protein+Crystals#No_fail_cryoprotection
. You may want to consider that it may not be the cryo that is causing
fracturing, but drop evaporation. (It's always worst this time of year,
when it's cold and dry.) In this case, the "no-fail" method (in which
the drops are left in the well as much as possible) or working in the
cold room (which raises humidity and slows evaporation) may be good
options.

Cheers, Roger Rowlett

On 2/4/2010 4:29 AM, Vellieux Frederic wrote:

Salve Claudia,

Glycerol is not the only cryo-protectant that is available. Have you
tried with ethylene glycol for example?

HTH (spero che aiuta),

Fred.

Claudia Scotti wrote:

Dear list,

I'm trying to freeze crystals in cryoconditions containing the
following:

0.1 M Sodium acetate pH 4.4

2.15-2.3 M Ammonium sulphate

7% n-butanol

15% glycerol

The problem is that the crystals (beautiful hexagonal prisms) seem to
shatter in a random fashion: some are unaffected, some, coming from the
same drop, disgregate miserably. One even split into three parts, of
which two disgregated and one survived perfectly well.

I've tried both by moving the crystals directly in the cryocondition
and by progressively increasing the glycerol concentration to no avail.

Shall I just select those that survive?

I was wondering if anybody has ever had this same problem and if
freezing under oil could be an alternative. If yes: any suggestions on
how to fish the crystals? I tried it in the past, but I found it very
difficult...

Experiences and suggestions are welcome.

Claudia

Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di
Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia
Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673

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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Freezing under oil
From: Jim Pflugrath Jim {- dot -} Pflugrath {- at -} RIGAKU {- dot -} COM
Date: 2010-02-04

Next message:
Subject: EMBO Course on Macromolecular Complexes: Grenoble, 31 May- 5 June 2010
From: Carlo Petosa carlo {- dot -} petosa {- at -} ESRF {- dot -} FR
Date: 2010-02-04