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

Matthew Connelly via (by MConnelly from
Fri Jul 24 03:16:51 EST 2009

When you freeze aqueous solutions slowly you form larger ice crystals. This would segregate water away from proteins and into the crystals of ice. As the amount of unfrozen water drops the protein becomes more and more concentrated and loses the water shell required to maintain its structure (remember the primary force for protein structure is the organisation of hydrophobic and hydrophilic residues). This quickly leads to protein denaturation and aggregation, and is one of the reasons why freeze thaw cycles are so damaging to proteins. 

Proteins are frozen quickly to produce the smallest ice crystals possible as quickly as possible, which minimises the disruption described above. Formulation of the protein with excipients such as polyols and carbohydrates can also help dramatically improve stability during freeze thaw by helping to lower the total amount of water bound up in ice crystals, and to take the place of water at the proteins surface. This can allow you to freeze proteins more slowly without causing damage.

Not sure about cells, but I think that the freezing rates necessary for cryopreservation are actually dependent on the cell/tissue type...


-----Original Message-----
From: methods-bounces from [mailto:methods-bounces from] On Behalf Of Xuan Yang
Sent: 24 July 2009 03:25
To: methods from
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.


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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