EDIT: I KNOW IT ultimately RESULTS IN CELL LYSIS.
My question is how? Is it just crystal formation? It seems to me that snap freezing the cells results in a sensitization of sorts of the cell membrane (and at a different rate than the nuclear membrane), making the my cells easier to lyse with other downstream applications. But I've been having trouble finding literature on this.
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What is the effect of snap/flash freezing pelleted cells? Specifically the cell- and nuclear-membranes .
The pellet is not 100% dry, but rather has trace amounts of PBS from a previous wash step.
Overall it appears to be destabilizing the cell membrane (guessing this is the same mechanism as if done in liquid: formation of ice crystals).
But what is the effect on the nuclear membrane?
Thanks Arslan, but my question is regarding what is happening to the membranes during the freezing process - e.g. when used as part of a cell lysis method.
Hi Lauren,
I have not done it as a routine method but it takes few freeze/thaw cycle to disrupt both cell and nuclear membrane so as to get a lysate that can be used for downstream processing. However, few times I have tried it..the protein yield was much less when you compare it to kit such as NE/PER from thermoscientific which works great. Hope this helps.
Thanks Anil, but I'm trying to understand what's actually happening to the membrane during the snap-freeze that makes the cells easier to lyse with downstream applications. It seems to me that there has to be something more than just crystal formation.
For my method, I snap-freeze my cells once to prepare them for subsequent isolation steps. When I snap-freeze my cells first I get a clean organelle separation (done using specific lysis buffers downstream). But if I skip the snap-freeze I don't get a clean fractionation.
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