Re: Freezing

["S.Ravichandran" <ravi@cmb2.saha.ernet.in>]

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>On Thu, 23 Sep 1999, Kunchithapadam Swaminathan wrote:

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> Dear :
> 
> I have got a crystal and the condition is:
> 
> 100 mM Tris-HCl, pH 7.5
> 150 mM Nacl
> 20 mM  Ca Cl2
> 
> (The crystal dissolves in conventional method of freezing, time course
> transfer in artificial mother liquor + 5%, 10%, 15%, 20%, 25% glycerol, MPD,
> PEG400).  I am looking for suggestions/advice for freezing crystals grown in
> pure salt environment.  Any tips?
> 
> With regards,
> Swami
> 
>   ****************************************************************************
>   Dr. Kunchithapadam Swaminathan		Phone: 	(65) 873 2173 (H)
>   Institute of Molecular Agrobiology			(65) 872 7408 (W)
>   The National University of Singapore		Fax:	(65) 872 7007 (W)
>   1 Research Link, Singapore 117604		Email:	nathan@ima.org.sg
> 
>   Web:	www.ima.org.sg/research/struct_bio/struct_bio.html
>   ****************************************************************************
> 


Hi Swamy,

Well, crystals that are grown in salt environment are commonly replaced 
with cryoprotectants such as glycerol, ethylene glycol, MPD etc. If the 
salt concentration is too high, then there are ways to replace the salt 
with an equivalent 'high solubility' salt (say ammonium sulfate to 
ammonium acetate; see Fink & Petsko (1981)in Adv. Enzymol. Relat. Areas 
Mol. Biol. 52, 177-246 ; Wierenga et al. (1992) in J. Cryst. Growth, 122, 
231) prior to the addition of cryoprotectants.

	But in your case, as I understand that the crystal gets 'dissolved' 
in these cryoprotectants, when you are trying to replace it. Did you vary 
your soaking periods? Have you tried 'immiscible' oils instead of 
cryoprotectants? 

Very recently, there is a article in J.Appl Cryst regarding the usage of 
oils+cryoprotectants (Kwong & Liu, (1999), J.Appl. Cryst 32, 102-105) and 
the succesive one about the procedure of glutaraldehyde cross-linking by 
vapor diffusion method (Lusty, (1999), J. Appl. Cryst 32, 106-112)-  may be 
an useful reading. 

For other reviews, may be Garman & Schneider (1997)in J. Appl. Cryst. 30, 
211-237 and Rodgers (1997)in  Methods in Enzymology 276, 183-203 will be 
helpful.

Cheers,
Ravs