Hi. I think if you freeze a aqueous solution slowly you will get pure crystals of water forming first that exclude any solute that doesn't fit into the lattice - effectively concentrating proteins into a smaller and smaller volume (freezing out). This is maybe why your proteins are precipitating. Flash freezing is too fast for the water to form pure crystals.

I believe the freezing out process is also used to make "ice beers".

Hi. I think if you freeze a aqueous solution slowly you will get pure crystals of water forming first that exclude any solute that doesn't fit into the lattice - effectively concentrating proteins into a smaller and smaller volume (freezing out). This is maybe why your proteins are precipitating. Flash freezing is too fast for the water to form pure crystals.

I believe the freezing out process is also used to make "ice beers".

Freezing temperature of pure water is higher than that of its solutions. This is why in some northern countries during the winter they spread salts on the roads to melt the ice.

Slow freezing of a protein solution leads to its separation to pure frozen water and liquid drops that contain upconcentrated protein. Plus you probably have other additives (like salts), that become upconcetrated during this process as well. If your protein solubility depends on the ionic strength, you will see precipitation due to the salting out effect.

Another thing is that all proteins undergo cold denaturation transition. If the temperature of this transition lies above the freezing temperature of your upconcentrated solution, you will observe protein precipitation associated with aggregation of partially folded conformations.

The above answers are quite good. I would question the relevance of cold denaturation in a system that has ice formation though. Cold denaturation relates to effect of temperature only (i.e. chill only - consider the thought experiment where you could supercool with no ice formation); add ice in the system, and the other effects listed above (cryoconcentration etc) become more important. Another factor is ice-surface induced unfolding of the protein and the selected storage temeprature (above or below Tg" of the matrix).

From the perspective of somebody who has to freeze 10s to 100s of Litres of protein solution and keep it stable for years, flash freezing is obviously not a possibility. The proper selection of buffer and cryoprotectant is key and suprisingly, the answers are not that diverse. Some references of possible tangential interest

Singh et al Pharm. Res. 28(4) 873-885 (2011) DOI: 10.1007/s11095-010-0343-z

Strambini GB, Gonnelli, M. (2007) Protein Stability in Ice. Biophys. J. 92:2131-2138.

Ok what about if you are out of liquid nitrogen and only have dry ice. What do people think? The rate of freezing is probably slower but will it make a big difference?

Although I've had better experience with unstable proteins by freezing them in liquid nitrogen, I prefer that method.

For small volumes (1 mL or so), not likely. Just be wary of CO2 getting into the container, (esp if stored on dry ice) and "acidifying" it unless you have a tight seal. And seal integrity will change (generally degrade) with low temperatures.

I think methanol and dry ice would be an adequate replacement for liquid nitrogen if you are in a pinch.

I thought this was an interesting observation since we slow freeze cell pellets for long-term storage. Thank you all for the interesting answers.

The above answers explain it.most of the reasons very well. Although we had good experience with freezing our enzymes in phosphate buffer (in contrast especially to Tris-HCL buffer--forming free base-- or some of the Goods buffers).

With cold sensitive enzyme It is also important to avoid a slow process of the reverse process i.e., thawing. We had several proteins which if we did not thaw them under hot water and gentle shaking, they became inactive due to a time dependent unfolding process at low temperatures. Beside the increase in protein conc and thus precipitation / salting out effects / pH changes etc the folding /unfolding/changes of fold of proteins due to low T are other reasons for flash freezing and fast thawing.