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

Matthew Connelly via (by MConnelly from
Fri Jul 24 05:19:25 EST 2009

I don't know whether cryostability of the protein would help indicate ease of crystallisation, as you are to  some extent altering the nature of the protein by crystallising it, whereas in freezing you are trying to avoid modifications to the structure for preservation purposes. However, you are right that freeze concentration and suparsaturation sound like similar processes, so there may be a corollary between protein stability on freezing and how closely the crystallised structure reflects the native protein structure. After all, both reflect the proteins tendency to resist structural changes in low water environments. Unfortunately I am no expert on protein crystallography so I have no idea how likely that is!


I must say I'd never thought that cells might be easier to store frozen than proteins before, that is very interesting!


From: Xuan Yang [mailto:pattisyang from] 
Sent: 24 July 2009 11:05
To: Matthew Connelly
Cc: methods from
Subject: Re: Confused about the different principles to freeze & thaw cells and purified protein


Dear Matt,


Thanks for the great & indepth explanation! You must be very good at physics.


Interestingly, it was obvious that some proteins were born tougher than others and could tolerate freezing and thawing process much better. Since the freezing process seemed like a process of supersaturation, I was wondering whether a better tolerance would indicate a better chance to form crystals.


Moreover, Prof. Terese M. Bergfors mentioned in the book "Protein Crystallization" that "Cells or bacteria tolerate freezing (-70ºC) better than many purified proteins", as a result, it would better to store expression hosts rather than purified proteins. And since cryopreservation was cell/tissue type dependent, I was wondering whether anyone would like to share some nice strategies to store expression hosts, especially E.coli. 


Thanks in advance!




Xuan Yang

2009/7/24 Matthew Connelly <MConnelly from>

	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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