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Sonication issues! - (Mar/01/2010 )

Following sonciation of my cells, I centrifuged as normal to harvest the insoluble protein fraction. Only to find I couldn't get a significant pellet no matter how much I spun the samples down!

Any ideas? Perhaps the sonication time was excessive?


How long did you sonicate, and did you have the sample on ice?

-lab rat-

What buffer did you sonicate the sample? If the insoluble protein is extracted by the buffer, I guess you might see any pellet.


I always sonicate at a 1:3 ratio (5 mins actual sonication time, 15mins off) per pellet from 200ml of culture. My samples are always kept on ice during the procedure too. The amplification was 40%.
I was trying two different buffers, one was something I have always used (tris, DTT, EDTA and NaCl at pH 8), the other was bought from Qiagen. But I didn't get a pellet for either once I had spun them down!

I've never had this problem before but I guess I will soon find out if this really matters soon enough!