Glycerol prevents freezing at -20C (at least at 50%) but not at -80C. I am not sure if 25% is high enough to prevent freezing at -20C though....When you want to store antibodies at -80C the use of glycerol is pointless at any concentration. I concur with previous comments about the detrimental effects of freeze/thaw cycles. These are to be avoided by storing aliquots. It is good practice to have one working aliquot at 4C in the presence of a preservative like sodium azide or gentamycin so you can use it on a daily basis without any freeze/thaw cycles.

Dan,

to stabilize antibodies normally sodium azide is used, 0.02% (w/v).

Please avoid multiple freezing thawing by aliquots.

First did you take the antibody in and out of the -80 rather than aliquot? That would be freeze thawing, which is bad for any protein. In my experience you shouldn't store antibodies at -80, unless lyophilized. Make sure your antibody is at least 1mg/mL and you can store at -20C aliquoted, once you remove an aliquot don't refreeze. Your 25% glycerol/PBS is a pretty good storage solution. But if you didn't do the above listed issues then maybe you do need to optimize the storage. Try a general PBS, 2% BSA with 25% glycerol which is usually sufficient, but if that doesn't work you can then try an additional 0.05% tween-20 (also, look into sucrose, trehalose, DTT, etc.).

For many antibodies, freezing at -20°C or -80°C in small aliquots is the optimal storage condition. Aliquoting minimizes damage due to freezing and thawing, as well as contamination introduced by pipetting.

The size of the aliquots will depend on how much one typically uses in the experiment. Aliquots should be no smaller than 10 µL.

Repeated freeze/thaw cycles can denature an antibody, causing it to form aggregates that reduce the antibody’s binding capacity.

Storing at -20°C should be suitable for most antibodies; there is no perceptible advantage to storing at -80°C.

Diluting antibodies to working concentration and storing at 4°C for more than 24 h should be avoided. Proteins in general are less susceptible to degradation when stored at higher concentrations, ideally 1 mg/mL.

Glycerol prevents freezing at -20C (at least at 50%) but not at -80C. I am not sure if 25% is high enough to prevent freezing at -20C though....When you want to store antibodies at -80C the use of glycerol is pointless at any concentration. I concur with previous comments about the detrimental effects of freeze/thaw cycles. These are to be avoided by storing aliquots. It is good practice to have one working aliquot at 4C in the presence of a preservative like sodium azide or gentamycin so you can use it on a daily basis without any freeze/thaw cycles.

 I do keep the antibody in aliquots, but I freeze in liquid nitrogen before store in -80C . I try to store the antibody in -20C to avoid freezing now .Thanks everyone!

 check your antibody's purity. the poor purity, the poor stability.

My experience has been that antibodies stored at -20C with significant amounts of glycerol to prevent actual freezing of the solution (50%) can retain their full activity for years (yes, aliquoted to avoid repeat freeze thaw cycles).  I suspect it is your initial freezing conditions that is damaging your protein, since liquid nitrogen is -210C to -196C at normal atmospheric pressure.  That is rather extreme treatment for an antibody and not really necessary.  Check out the antibody storage guide from abcam linked below.

http://www.abcam.com/protocols/antibody-storage-guide

Avoid freezing and thawing.

Normally, I keep antibodies at +4C for at least one month and keep the rest frozen at -70 without glycerol but with a concentration around 1 mg/mL. I thaw slightly larger fractions that lasts me for a month thus avoiding freezing in small fractions or repeated freezing and thawing.

Generally, a polyclonal antibody should be stable at +4C for years.

I normally store monoclonal and polyclonal antibodies that I purify at -20 °C in PBS containing 0.02% (w/v) Thimerosal (aliquoted). Glycerol can be added (20-50%).  I prepare myself also antibodies-HRP conjugates and I store them at -20°C in PBS, 1% bovine albumin, thimerosal and 50% glycerol (also in aliquots). In this manner, my purified antibodies are stable for up to 2 years.