sonication in immunoprecipitation - (Jul/12/2007 )

dear all
this is my first experience with immunoprecipitation, and I have a question:
can we use sonication to help achieving efficient cell lysis?
thanks

Yes, you can. However, you do run the risk of shearing proteins and protein complexes. The same problem exists when you are interested in large (non-complexed) proteins. The larger the protein or complex of interest, the more likely you are to experience a break somewhere within it when employing mechanical disruption. If you do sonicate, be conservative about it: perhaps 20" instead of a typical 2x20", for example. Syringe or fine tip shearing could also be used: they are a bit more gentle (and less effective, in my experience), but excessive processing could still lead to the same problems.

-Eric

ok, what about 2 cycles of freeze-thaw (- 80 to +4 degess)?
one more question, should I start IP immediately after preparation of the total cell lysate or can I store smaples at - 80 and then strat IP procedures later?
thanks