-20oC is not the best way to store proteins in a frozen state. It is not cold enough to prevent sublimation of ice at the surface of the solution, leaving dried sample. Also, frost-free freezers occasionally warm up to prevent frost from accumulating inside. If you have access to a -80oC freezer or liquid nitrogen, store your protein there. If not, consider storing it in an unfrozen state at -20oC with 50% by volume glycerol. It's a good idea to include some glycerol, such as 20%, in frozen protein samples as a stabilizer, in any case. Finally, when freezing the protein for the first time, freeze it rapidly by immersing it in liquid nitrogen or a dry ice-ethanol (or acetone) bath. Rapid freezing prevents separation of solutes from the ice.

Dear Dr. Adam Shapiro,

Many many thanks for your prompt suggestion. I have already completed this part of stability study by using batch wise increasing the percentage of stabilizer i.e. glycerol (from 10 to 50 %) and storing protein at -80°C. However, very little quantity of precipitate was visualized after three months at both temperature that is -20 and -80°C. Hereby I kindly request you to please suggest any alternatives for example i) changes in buffer composition ii) addition of anti-precipitating stabilizers iii) any other valuable suggestions which help me in solving the problem of precipitation.....

You confuse me. You say that "very little quantity of precipitate was visualized after three months" in your storage stability study using glycerol as a stabilizer. It seems from this that you have your solution already.

Some additional changes to your storage conditions could be made. The sodium azide isn't necessary to inhibit growth of bacteria, since the sample is frozen. The buffering is a bit weak at 20 mM Tris and might be increased to 50 mM. The salt concentration is quite high. If it is not needed to prevent aggregation, you might lower it to a more physiological 150 mM.

I'm not sure I follow the history very well but some interesting information might come from any characterization of this precipitate and the amount that forms.

Was there actual loss of total CRP mass or activity in the container?  Can the precipitate be isolated by centrifugation?  Sometimes the appearance of a precipitate looks worse than what has actually occurred in terms of amount (a lot of hydration).  If isolated, does the precipitate dissolve in the formulation buffer?  Can you analyse the precipitate on SDS-PAGE?  Will the clarified supernatant reform precipitate upon multiple freeze/thaw cycles?  Is affinity chromatography the only purification step? In other words are there traces of any contaminants?  I've seen trace contaminants as cryoprecipitates and that is actually a purification.  How did you derive the formulation buffer which is usually an extensive study?  How do you perform the thawing?  

Worse case is that you could try lyophilization and I believe CRP is stable to lyophilization, right?

Precipitation/aggregation may occur under two conditions:

1.Returning from deep freeze to room temperature is factor reported to cause precipitation/aggregation in some protein.

2. The molarity (concentration) of protein may also cause precipitation/aggregation since at high concentration  proteins are tend to  aggregate.

I am surprised by your inclusion of 5 mM CaCl2 in your buffer. I haven't worked with your protein, but unless it specifically requires Ca2+ for stability, this is not a good idea. Even much lower Ca2+ concentrations (and other divalent cations) can cause precipitation of protein in long term storage. We generally store proteins without divalent cations, but also with addition of some EDTA (usually not more than 1 mM), just to mop up any dvc that may have been introduced in the process of protein isolation. Anti-aggregatin osmolytes also can work well, but glycerol has a spotty history in that regard. ≥400 mM proline and high concentations of trehalose are suposed to be better, but I don't have personal experience with that; I have been lucky in that glycerol has worked will for my proteins (50%, with storage at -20 or -80 °C; glycerol also is a cryoprotectant, so it doesn't matter how fast you freeze your solution, there is no separation of water or solutes during freezing, even sowly, and consequeently no damaging ice crystal formation or local high concentations of solutes). In your shoes, I would get rid of the calcium and try a physiological NaCl concentration and an osmolyte (and probably some EDTA). Mohsin also makes a good point... if your protein is above some threshold concentaration (which can only be determined empirically), it will invariably aggregate, sometimes reversibly, usually not, so try a lower protein concentration. Any solute you introduce that interferes with downstream assays etc. can be easily removed by buffer exchange on a PD-10 column (Sephadex G25), or equivalent microfuge spin columns for micro amounts. Hope that helps.

Have a look at this, and maybe google some similar stuff:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1569150/

Three things:

1. Tris buffer is not the best choice if you have to freeze /thaw your protein: the temperature dependence of its pKa is one of the highest among common buffers. You risk to change the pH of your solution by 0.03 point by 1°C decrease (your pH may increase from 8.0 to 9.5 or even higher at -20°C or lower temperatures). 

Good, N.E. (1986) Biochemistry 5, 467

2. However, when I have to freeze protein samples, I ALWAYS do it in liquid nitrogen, because proteins precipitation usually is minimized by fast freezing.

3. If you want to find ptimal conditions for protein storage, use Differential Scanning Fluorimetry, using a matrix of experimental conditions in the presence of Sypro Orange

Niesen et al., Nature Protocols 2, 2212 - 2221 (2007) .

1)  DMSO was the only successful cryoprotectant of several I tested for preservation in l-N2 of function of purified eIF-2 (euk. init'n factor 2).  (note: vital for cryopreservation of human embryos).  

2)  nondetergent sulfobetaines remarkably stabilize proteins against aggregation.  (Anal Biochem. 352 (2006) 299–301)

(a zoo of other stabilizing 'compatible solutes' including ectoine, glycoin, trehalose, trimethylamine-N-oxide, etc are also protein stability research tools)

Actually I cannot add any news to the list of suggestions above. I can tell you only as much as I have no problem of precipitation of my highly purified protein (a detergent-solubilized trans-membrane protein of 25 kDa) when I store it 

- at -80 °C in deep-freezer,

- in 150 mM NaCl, 50 mM K,Na-phosphate buffer, pH 7.0,

- in the presence of 10% (v/v) glycerol,

- in the presence of detergent at 1.5 x c.m.c. (you do not need this, of course), and

- under nitrogen in the cryovial.

Good luck to find the optimal conditions for storage of your purified protein.