If you are asking about short peptides without defined tertiary structure, there should not be any degradation caused by freeze/thaw cycles. If you are asking about proteins, the answer depends on the protein, but in my experience most proteins handle repeated freezing and thawing well, as long as the freezing is rapid, as you would expect it to be with liquid nitrogen.

Freeze the protein in -80 conditioner or liquid nitrogen repeatedly is actually a method to test the protein stability, where unstable protein may quickly break apart within several freeze-melt rounds. But it's hard to say if that harms your sample, since you've only drowned it 3 times. 

Dear Michael, thanks for your attention, well I'm concern about stability of my peptides after this,  but the fact is I freeze my sample at -196°C  and then  thaw it at 30°C after freeze, and i did this process for three time... so you think for sure despite of these degrees and repeat(3 time), my peptides are intact?

Thank you for your rating. I can't guarantee that the peptides remain intact, but I think it is likely to be. 

I haven't performed this freeze-thaw experiment myself, but I heard of that from our collaborative lab, and I remember that they repeated tens of rounds to semi-quantify the protein stability. 

If you are asking about short peptides without defined tertiary structure, there should not be any degradation caused by freeze/thaw cycles. If you are asking about proteins, the answer depends on the protein, but in my experience most proteins handle repeated freezing and thawing well, as long as the freezing is rapid, as you would expect it to be with liquid nitrogen.

Hi Ruhollah...I vote for the answer from Adam. In my experience, a rapid cooling should save the protein at least for 3 - 5 cycles. However, it depends on the protein. Some proteins can be thermo-sensitive.

How do you normally store peptides under which conditions.

If you have them in Acetonitrile they will not really freeze in a -20 C freezer. You will have a cold solution were your peptides can stick to the wall of the container. Then snap freezing is better, 50% Acetonitrile in water will stay frozen below roughly -20 C.

If you have salt or buffers in your sample, putting the sample just like that in a a -20 C freezer is not good. The pH of certain buffers will change upon freezing dramatically, result can be damaged protein.

Peptide stocks I usually would store them in small aliquots in a -20 or -80 freezer but snap frozen in liquid nitrogen, wil take about 15-30 sec wile freezing in a -20 or -80 will take up much longer.

And yes there are proteins who really do not like the frozen state or were you can only do that one time (freezing and thawing).

I agree that it depends on the type of protein. In general cryoprotectants such as sucrose and trehalose will help in protein from freeze thaw streess

Dear Ruhollah Vasegh,

You got quite a bit of sound experimental advice. I only try here to point out some of the more theoretical underpinnings of the above answers. In the last 20 years, or so, the physical understanding of the protein structure became to be understood on a deeper level. We now believe that the protein structures are unique because of the intricate dynamical coupling with the surrounding solvent. In actuality proteins form different glassy states that glassify at different temperatures. Usually, interior is formed from a glassy state (a disordered atomic state) that is solid around room temperatures (and liquifies at temperatures in excess of 50C), the surface residues couple with the solvent and form a glassy state that is liquid down to around 200K and then solidifies (glassy transition temperature) and the remaining third glassy system is formed by the solvent itself that solidifies at highly variable temperatures depending on the content (salts, sugars etc) but usually below 200K.

As a consequence rapid freezing (crossing the glassy transition temperature Tg) solidifies the protein and stops protein activity at around liquid nitrogen temperature, and as such is a very positive event. Slow freezing creates rapid growth of water or more general solvent crystals that usually leads to the damage of a natural protein structure.

Usually, recovery from such a situation is not reversible, and is equivalent to "making scrambled eggs". For short peptides that usually have very rudimentary tertiary structure or not at all, it means that you can safely repeat freeze thaw cycle many times before they get damaged (but mostly by side chemical reaction that accompany physical changes to the system) much less.

In essence do not worry about your peptides (toxins are usually well folded), but nothing substitutes experimentation, so you should perform some kind of a functional assay after every cycle of freezing. Good Luck.

 it depent on the nature of protein