I totally agree with Elke. Urea solutions should be prepared fresh and in smaller batches. Of course the components of your rehydration buffer will stay stable at a storage at -20°C but Urea solutions form crystals in freezed condition and you need to heat it to resolve your rehyd-buffer for usage. However, it is not recomented to heat urea solutions because urea tends to break down to isocyanate spontanously and this reaction could be accelerate by heat.

Isocyanates can lead to carbamylation of proteins which could be seen as additional (artificial) spots on the final 2D gel and could impaire further MS analyses of these spots. When we use urea for solubilization- or rehdration-buffer we remove existing isocyanates with ion exchange resin (like AG 501-X8 resin from Biorad) before we make the final Buffers.

I use the same recipe as yours, but without ASB-14.

I've seen gels improve dramatically after changing to a fresh batch of rehydration buffer just this week. The old batch had been in the freezer for 6+ months, the new batch was made with the same chemicals.

Bottom line for my lab: make smaller batches of rehydration buffer, discard after a few months, err on the side of caution. This is certainly not the most expensive part of your 2D experiment, so make fresh whenever feasible to save yourself some serious time and frustration.

I totally agree with Elke. Urea solutions should be prepared fresh and in smaller batches. Of course the components of your rehydration buffer will stay stable at a storage at -20°C but Urea solutions form crystals in freezed condition and you need to heat it to resolve your rehyd-buffer for usage. However, it is not recomented to heat urea solutions because urea tends to break down to isocyanate spontanously and this reaction could be accelerate by heat.

Isocyanates can lead to carbamylation of proteins which could be seen as additional (artificial) spots on the final 2D gel and could impaire further MS analyses of these spots. When we use urea for solubilization- or rehdration-buffer we remove existing isocyanates with ion exchange resin (like AG 501-X8 resin from Biorad) before we make the final Buffers.

for references of isocyanate removal from urea see:

Busse, W. and Carpenter, F. H., Biochem., 15, 1649 (1976) or

Edelstein, C., et al., Biochem., 15, 1262 (1976); or

Spieker, H. and Polet, H., J. Biol. Chem., 251, 987 (1976) or

Traugh, J. A. and Porter, G. G., Biochem., 15, 610 (1976).

Thanx to everyone for your answers. I will probably prepare a new smaller batch of rehydration buffer and keep it at -20oC and use it for my experiments.

You haven't mentioned the types of strips you use.

My recepie for rehidration buffer for strips pH 3-10 is:

8M Urea, 2M Thiourea, 2% CHAPS, 1.2 % (v/v) DeStreak Reagent, 0.5%(v/v) IPG-Buffer pH 3-10, Trace of Bromophenol blue

As for the strips pH 4-7 I use the following buffer:

8M Urea, 2M Thiourea, 2% CHAPS, 10mM DTT, 1.2% IPG-Buffer 4-7, Trace of Bromophenol Blue

It is recomendable to prepare fresh buffers. However, I keep them at -20oC for up to 6 months and I haven't experienced any problems so far.

Hope that this helps...

Regards

Hi Alexandros,

I use 8M urea, 2M thiourea, 4% CHAPS; broad range ampholytes and protease and phosphatase inhibitors. I aliquote my buffer and store it at -80oC. Works pretty well; no problems so far.