Andreas, you probably know all this, but I only understood quite recently. What happens is that as ice crystals form you get "brine rejection", the same thing that happens in the arctic when sea water freezes. Therefore you can have protein concentrated in pockets of high salt. Fine for some proteins, but others don't like it. And it can happen during (slow) thawing as well as during freezing. - Patrick
On 29 September 2014 16:02, Andreas Förster <docandr...@gmail.com> 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 > > > > > -- > Andreas Förster > Crystallization and X-ray Facility Manager > Centre for Structural Biology > Imperial College London > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36