Perhaps your protein would be less prone to precipitation if it were diluted. You could also try using a different pH or changing the salt concentration.

In any case, the purpose of flash-freezing is to avoid the separation of solutes from the ice that happens when it is frozen slowly, so speed is important.

A simple way to flash-freeze proteins is to make small aliquots in microcentrifuge tubes, such as 100 µl, then insert the tubes into a layer of powdered dry ice. I prefer this to using a dry ice-ethanol or dry ice-acetone bath because it avoids solvents that remove the labeling from the tubes. Be sure to wear eye protection when bashing dry ice pellets to powder them. I use the top of a claw hammer on dry ice in an ice bucket.

You can also put the tubes in one of those cardboard boxes with a grid of dividers and pour liquid nitrogen into it. With both methods, after a few minutes, the protein aliquots will be frozen solid and can be stored at -80oC.

Thaw the protein slowly on ice or quickly in the hands. Since your protein seems to like to precipitate, quick thawing is probably better.

Storing the protein with 10% glycerol will help it to thaw faster and can also act as a stabilizer.

Dear Deepak,

if you are lucky you can just quickly put small aliquots of your protein from the fridge in some volume of liquid nitrogen, take it out after a minute and subsequently store it at -80 °C for many months or even years. This is possible in many cases. However, this also widely depends on the protein and its structure. In some cases, additives like a few percent glycerol or a reducing agent can help to prevent aggregation by freezing or dilution of the protein helps. Some people also try uncommon strategies to avoid freezing at all. I suggest to test freezing with a small quantity first and design a quality control experiment, which shows with some confidence that folding and oligomeric state are really still fine in a specific optimized buffer after thawing. This could be some kind of spectroscopy or native gel electrophoresis or light scattering to test the protein and ensure reproducibility of crystallization trials. Thawing should in my experience just be rather slow and under consistent conditions. Good success!

Perhaps your protein would be less prone to precipitation if it were diluted. You could also try using a different pH or changing the salt concentration.

In any case, the purpose of flash-freezing is to avoid the separation of solutes from the ice that happens when it is frozen slowly, so speed is important.

A simple way to flash-freeze proteins is to make small aliquots in microcentrifuge tubes, such as 100 µl, then insert the tubes into a layer of powdered dry ice. I prefer this to using a dry ice-ethanol or dry ice-acetone bath because it avoids solvents that remove the labeling from the tubes. Be sure to wear eye protection when bashing dry ice pellets to powder them. I use the top of a claw hammer on dry ice in an ice bucket.

You can also put the tubes in one of those cardboard boxes with a grid of dividers and pour liquid nitrogen into it. With both methods, after a few minutes, the protein aliquots will be frozen solid and can be stored at -80oC.

Thaw the protein slowly on ice or quickly in the hands. Since your protein seems to like to precipitate, quick thawing is probably better.

Storing the protein with 10% glycerol will help it to thaw faster and can also act as a stabilizer.

It probably would be a good idea to flash freeze your protein in a buffer containing 20 v/v% glycerol. Label a cardboard box not just the lid with the date and a specifications regarding your protein. I normally just dilute the protein with the glycerol buffer and subset that into small 20µL aliquots and flash freeze the protein in liquid nitrogen in a metal container for a minute, and then remove the tubes of flash frozen protein with a large slotted metal spoon to transfer to a labeled cardboard box, and immediately put that in the -80°C freezer. Make sure your store your protein at sufficient concentration and in small aliquots because refreezing a single aliquot of protein is bad idea for protein function.

Glycerol is good for freezing your protein because it is a cryo-protectant that blocks ice crystal damage. But when you are ready to take some aliquots of your protein out of the -80°C just thaw it on ice. It will thaw pretty quickly.

Then when you are ready to crystallize your protein, Remember that it is in glycerol and that glycerol to crystallographers is an antinucleation agent. (That means it inhibits the nucleation of protein crystals. Which is only good if your protein crystallizes immediately but imperfectly and you want your protein to crystallize slowly and in a better configuration. If your protein doesn't easily crystallize, glycerol will just make your life such trouble.) You should exchange out the glycerol in your sample for a new buffer your protein is soluble in without glycerol. you could do that by dialysis or a spin filter or even by size exclusion with a new buffer. But don't do size exclusion because your protein will be even purer but so much less concentrated.

Thanks for sharing the detailed explanation to all the scientists.

I got the answer to what I was looking for, I will try with a small volume in different aliquotes quickly. Thanks a lot once again