Fwd: Confused about the different principles to freeze & thaw cells and purified protein

[Xuan Yang]

---------- Forwarded message ----------
From: Xuan Yang <pattisyang from gmail.com>
Date: 2009/7/24
Subject: Re: Confused about the different principles to freeze & thaw cells
and purified protein
To: Zhonglin Chai <Zhonglin.Chai from bakeridi.edu.au>


Dear Mr or Ms Chai,

Thanks for the reply and based on your explanation, the key difference if
not the only difference should be that cells have fragile membrane while
purified proteins do not. Slow freezing with DMSO (DMSO is critical and
thanks for reminding me of that) should minimize the damage to cell
membrane, while rapid freezing with glycerol should maximally retain the
activity of purified proteins. Since I have no idea about what happened in
the process of freezing and thawing, which seemed like an independent and
mysterious filed itself, the exact reasons underlying these processes
remained elusive to me.

Then I did a little google about freezing and thawing. Based on a
literature titled "Protein Denaturation during Freezing and Thawing in
Phosphate Buffer Systems: Monomeric and Tetrameric β-Galactosidase", at
least pH change was involved in the process of freezing and thawing.
Moreover, "thawing fast" seemed like a general principle applied to both
cells and purified proteins. Thus if the protein sample was not heat
sensitive, thawing on ice should be disfavored.

Sincerely,

Xuan Yang
2009/7/24 Zhonglin Chai <Zhonglin.Chai from bakeridi.edu.au>

My personal view on this is that for cells, we tried to avoid formation of
> ice crystals which break cell membrane to kill your cells. This is achieved
> by the protocol you described with slow freezing (in DMSO) and fast thawing
> in 37oC degree water bath. For proteins, particularly for enzymes, or other
> bioactive samples, they may be sensitive to high temperatures, the best way
> to thaw is on ice to avoid exposing them to an undesirable higher
> temperature such as room temperature.
>
> Z Chai
>
> -----Original Message-----
> From: methods-bounces from oat.bio.indiana.edu [mailto:
> methods-bounces from oat.bio.indiana.edu] On Behalf Of Xuan Yang
> Sent: Friday, 24 July 2009 12:25 PM
> To: methods from magpie.bio.indiana.edu
> Subject: Confused about the different principles to freeze & thaw cells and
> purified protein
>
> Dear Sir or Madam,
>
> When we store cells (especially eukaryotic cells), storage box filled with
> isopropyl alcohol was used to prevent the temperature from dropping too
> fast. While thawing the cells from liquid nitrogen, we put the tube
> immediately into 37ºC water bath. Freezing slowly, thawing fast. This
> strategy was totally contrary to the way we deal with purified proteins,
> namely freezing rapidly (favorablely in liquid nitrogen), but thawing slowly
> (favorablely on ice). It was quite confusing and I was wondering whether
> anyone would be so kind to offer me some explanations.
>
> Sincerely,
>
> Xuan Yang
>
> National Laboratory of Biomacromolecules and Center for Infection and
> Immunity, Institute of Biophysics, Chinese Academy of Sciences, Room 1617,
> 15 DaTun Road,Chaoyang District, Beijing, China, 100101
> Tel: 86-10-64884329
> We will either find a way or make one.
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