sedimentation of cytosolic proteins - (Aug/02/2012 )

I have this recurrent problem with cytosolic protein extraction. The initial extract has all the proteins nicely solubilized, but upon storage at -20 over a period of time, a pellet forms, that doesn't dissolve upon thawing/vortexing. I have had some protein bands completely disappear due to this, and I think it's because some proteins are precipitating.

Has someone faced a similar problem? If so, could you please help me out?

Depends on the method of extraction and the buffers you use to keep them in. I use a digitonin extraction method and avoid using Dounce homogenizer. And I always keep my protein samples at -80C, not at -20C. And may I know what protein you are looking at in the cytosolic fraction?

some proteins can't withstand freezing. for this reason we add glycerol (to 50%) to extracts to prevent freezing at -20C.