Hi!

The storage of protein samples at -80 is always a critical step, because there is no way to predict the behavior of the protein (if, after freezing and thawing, it's going to be still stable).

Flash freezing in liquid nitrogen or addition of glycerol (or a combination of them) are common strategies that aim to prevent or limit protein damages, in order to keep them soluble and, hopefully, still active. Below you can find a link to some general guidelines that may help you. Most of the time they work very well, sometimes they don't...the only way to figure it out is to test them, and eventually adjust the conditions in order to find a good match that makes your samples "happy".

Storage conditions may also change according to the future use of the samples. For example, if you need to perform SDS-PAGE you can think about denaturing a small fraction and store it at -80 (or -20) directly in the loading buffer. Denatured proteins may be stored in this way indefinitely (as far as I know).

For chromatography, storage conditions are much more critical. 50% glycerol should work pretty well. The major problem is that the solution is highly viscous and if you inject a sample as it is on a column, you risk to damage it (because of the high pressure it generates). My suggestion is to dilute your sample before the injection, in order to decrease the concentration of the glycerol (5-10% should work, but you can check the datasheet of the column you plan to use to be sure about the concentration it can handle...).

A more general advice, after you thaw your sample, is to centrifuge it...this helps in removing particles that are aggregating or precipitating, which are not always immediately visible.

Hope I was helpful! 

https://www.embl.de/pepcore/pepcore_services/protein_purification/storage_purified_proteins/

Just want to add second what Patrizia wrote about chromatography.  Protein chromatography is hard, and you don't want to make it any harder than it needs to be.  Having a large amount of glycerol in there will certainly make it harder.  Use it only if you're sure that your protein needs it.  Most proteins are totally fine with a single unprotected freeze-thaw cycle, especially when they're still in crude lysate form. 

I agree with Patrizia's comments with one exception. After you thaw your sample it is better to take aliquots for your SDS-PAGE directly from your lysate without prior centrifugation (because you don't know if your protein still in solution or it's not soluble anymore). Then you add 2X SDS-Sample Buffer (under reduced and non-reduced conditions), centrifuge the samples at ~ 30,000 g, take the supernatant and load to the gel. In that case, the chance that you might loose your protein as a precipitate after just thawing will be much less. Good luck ! 

Thank you for valuable suggestions!

Another question, interms of stability of the protein in my case do you think it is better to store as cell (not sonicated) or as protein extracts for long term storage? I want my proteins to stay intact in their correctly folded form and they should keep their activity..

It would be a better idea to store cells rather than storing the protein extracts.

I see people have provided great suggestions to your question. I'd like add a little thoughts: 1) yes, storing your samples as intact cells is better than as extracted proteins if you prefer to keep their activity; 2) you may make several aliquots before freezing, this way later you just take one aliquot a time without risking the rest samples to freeze-thaw cycle; and 3) Given that you plan to run your samples after quite a long term storage, it may be a good idea to have a test for the stability after freezing. So you may have a few sets of aliquots, each set undergo a different freeze setup (buffer, glycerol concentration, etc...). After freezing them for one week, take one aliquot from each set and run the experiment that you plan for the future and see how the result is. An acceptable result is a good sign that your proteins have survive the original freezing procedure (as far as your experiment concerns) and are most likely remain that way over a long term storage under -80 degree.

Hope this is helpful and good luck!

Since you have sonicated your bacterial culture already, I suppose it is too late to consider freezing the cells. One good reason for adding 50% glycerol and freezing is that some enzymes lose activity when the solution is frozen. When dealing with such an enzyme, I have had good luck by adding 50% glycerol and freezing at -20 deg C, not -80 deg C. The glycerol allowed me to store the enzyme at -20 deg C while keeping it in solution (i.e. water in the liquid state). Note that the enzyme assays could be done with the enzyme in 50% glycerol solution. If you want to apply a technique that cannot be done when your proteins are in 50% glycerol, note that it will be difficult to do so. You would be better off making sure that you concentrate your proteins down before storing in 50% glycerol so you can reduce the glycerol concentration before the application by dilution as suggested above. 

Amazing answers much appreciated!! Thank you all.

  The other way you could do is  dialyze the   protein cell debris mix in  the appropriate buffer containing 50% glycerol. After dialysis volume would come down and then you could store the sample in  small volumes in -20. When you require  just dialyze the sample in the appropriate buffer without glycerol  with 3-4 buffer changes  and then the glycerol could be eliminated. The sample could be used for further analysis and down stream processing. 

There are not general rules for proteins so you can expect any result. I am afraid yu have to try with your own simple and compae with fresh prepared material. De-frosting is also a critical step and we do not realice that. Do it as quick as posible even heating a litle bit. I do not think glicerol will affect. It is employed in the simple buffer in order to increase the density of the simple. As I said, you should confirm the results experimentally. To run a samall SDS-PAGE do not take long.