[3dem] Precipitation observed after making freeze substitution cocktail containing 5% water

[Amar Parvate]

Hi  Qiaohui
I would agree with the 7-8% stock concentration for UA. Even at that
concentration, it would take a while to dissolve in methanol. Additionally,
I would filter it with a 0.22 micron filter, make aliquots and then freeze
in LN2. Dont know if LN2 was overkill but i have never seen
cloudy precipitation in my FS mixes and i was using 3% OS and 0.5% UA along
with some TA and glutaraldehyde.

Hope this helps!

Best


On Thu, Dec 19, 2019 at 11:31 AM Christopher Peddie <
Christopher.Peddie at crick.ac.uk> wrote:

> Sorry, I should have been more specific: 20% in methanol.
>
> ------------------------------
> *From:* Gibson, Kimberley <kimberley.gibson at yale.edu>
> *Sent:* 19 December 2019 15:08
> *To:* Christopher Peddie <Christopher.Peddie at crick.ac.uk>; Qiaohui Yang <
> qiaohui.yang at ymail.com>
> *Cc:* 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
> *Subject:* Re: [3dem] Precipitation observed after making freeze
> substitution cocktail containing 5% water
>
> It's not possible to make a stock solution of 20% Uranyl Acetate. Even
> making a 10% UA stock is not possible. At best, the solubility of UA is
> around 7-8 % in water. It's best to make a 5% stock solution, dissolved in
> water, for example, and add the quantity of 5%UA to the acetone. The
> precipitation of UA is likely due to the acetone as UA is not really
> soluble in acetone.
> Best,
>
> Kimberley Gibson
>
> Research Associate, Charles Sindelar Lab
>
> Dept. Molecular Biophysics and Biochemistry
> Yale University
> ------------------------------
> *From:* 3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Christopher
> Peddie <Christopher.Peddie at crick.ac.uk>
> *Sent:* Thursday, December 19, 2019 03:56
> *To:* Qiaohui Yang <qiaohui.yang at ymail.com>
> *Cc:* 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
> *Subject:* Re: [3dem] Precipitation observed after making freeze
> substitution cocktail containing 5% water
>
>
> Hi Qiaohui,
>
>
>
> Mix the osmium, water and acetone and cool to -20oC. Then add the uranyl
> acetate from a stock e.g. 20%. Cooling the mixture in my experience stops
> the uranyl acetate from precipitating out.
>
>
>
> Best,
>
> Chris
>
>
>
>
>
> *From:* 3dem [mailto:3dem-bounces at ncmir.ucsd.edu] *On Behalf Of *Qiaohui
> Yang
> *Sent:* 19 December 2019 08:39
> *To:*
> *Subject:* [3dem] Precipitation observed after making freeze substitution
> cocktail containing 5% water
>
>
>
> Hi everyone!
>
> We recently tried to make freeze substitution cocktail consisting of 1%
> osmium tetroxide, 0.1% uranyl acetate and 5% water but observed that the
> mixture turned slightly cloudy within 1-2 minutes. We tried to see if the
> precipitation would occur in the absence of osmium tetroxide, and we
> observed some cloudiness too after mixing uranyl acetate solution in water
> with pure acetone.
>
> Just wondering if anyone has encountered similar problem and if so, would
> appreciate some tips on how to overcome this problem.
>
> Thank you!
>
>
>
> Best regards,
>
> Qiaohui
>
>
>
> The Francis Crick Institute Limited is a registered charity in England and
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> 06885462, with its registered office at 1 Midland Road London NW1 1AT
>
> The Francis Crick Institute Limited is a registered charity in England and
> Wales no. 1140062 and a company registered in England and Wales no.
> 06885462, with its registered office at 1 Midland Road London NW1 1AT
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-- 
Amar Parvate
Saphire Lab
LJI
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