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I have experience with some proteins that don't tolerate freeze-thawing very well. It's hard to say exactly what the physical chemistry of this is, but it probably relates to (1) aggregation due to high concentration or protein or salts during the freezing process as water is removed, and/or (2) pH shifts due to changes in pKa of buffers/proteins as the temperature is lowered. Usually freezing in whole cells is less problematic than freezing purified protein solutions, but there are no absolutes. One protein we worked on could only be stabilized from cradle to grave in 20% glycerol, 100 mM DTT, and 4 deg C. Would not tolerate freezing, ever. Not even in cell pellets. Died at 25 deg C in a couple of hours--had to work quickly to do kinetics. Worst...protein...to work on...ever. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask] On 9/29/2014 11:02 AM, Andreas Förster wrote: > Dear all, > > I've encountered people who refuse to freeze cells and always lyse > fresh pellets. Better protein, they say. I've never had reason to do > so myself, or even to believe in their voodoo. Up until now, maybe. > > My protein expresses well and is almost all in the soluble fraction in > an expression test from a fresh pellet. The large-scale expression > from the same pellet, now frozen and thawed, yielded 90% insoluble > protein. > > If it's the freezing that dooms the protein, I'm happy to redo the > fermentor run. Are there other examples out there of this? > > Thanks. > > > Andreas > > > >