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?
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!