Sonication query - How long do people sonicate samples? (May/14/2005 )
Hi, just a general query: how do other people sonicate samples after protein overexpression? In one lab a postdoc divided samples into 5ml or bigger pots and sonicated on maximum for for 20 seconds with a minutes between each sonication 2-3 times (on ice). In my present lab people put their whole sample in one 50ml pot and sonicate for longer e.g. 30-60 seconds and more times e.g. 4-5. Is one way better than the other, is it even important as long as the cells change consistancy and lyse completely and do not warm up? I have started sonicating in 1 big pot for 20 seconds with 1-2 minute intervals as in the method above I think the sample starts to heat up. No-one I have asked seems that sure about this, I was wondering if anyone on the forum does...?
I use 15 sec for 2mins interval stop 15 sec. The total volume for sonication is 5ml. and it seems ok.
I use 10 10s bursts (microtip, 50% amplitude) with 15s pause between each burst for a 40 ml suspension of cells (E. coli, from 2 l of culture, OD600 approx. 2).
As long as the suspension does not wark up is OK, but be careful - I have read that sonication may do some damage to certain proteins.
Sonication results in extensive heating for the sample and thus results in protein denaturation. You may see this effect as foaming on top of your sample as indication of denaturation. Therefore it has to be done on short intervals and then incubate on an ice cold bath between each sonication cycle. Good Luck!