Sonication mysteries - (Mar/08/2010 )
Hello!
I'm in the "getting organized" phase of a secretome analysis on some mouse middle ear epithelials. The plan is to treat them with NTHi lysates, which I'm in the process of making. The problem that I'm coming across is that my predecessor, in what I've discovered is typical fashion, has left exceedingly poor notes on protocol for making these lysates. I've grown the bacteria -- fine. I need to sonicate them now to release the assorted proteins, but the protocol says only "sonicate bacteria" -- no indicator of power/duration/anything. I'm not having much luck searching for guidance on the internet, and no one here in my lab seems to have any clearer idea.
Any thoughts from you? I've sonicated something maybe twice in my life, so I don't even have prior experience from which to make any conclusions.
Isn't there a way to contact that person to ask for clarification? It would be a better choice rather than looking around for answers in the net. Drop that person an email or call if need be.
Alternatively, you'll just have to optimize the settings, which will help you get familiar with the optimization steps, BUT this will take away your time.
Google "NTHi sonicate" you'll get a couple thousand hits with protocols and papers mixed in with materials & methods for your cells. 
Ah, see -- I just wasn't googling the right things. God knows why I didn't think of "NTHi sonicate" before.
As for contacting the original person, the problem comes about that my predecessor was unreliable and disorganized when they were actually working this position... they're impossible to find/get information from now.
For references' sake, I ended up taking a 10 uL sample and diluting into 1 mL PBS, taking the OD 600, and sonicating in 15-second bursts at 40% power until the OD dropped by about 60% -- that seems like it will be sufficient for our purposes, as I'm wary of doing it for longer since I obviously don't want to risk denaturing all the assorted proteins we're trying to expose our cells to. Does that seem reasonable?
clickpopclick on Mar 9 2010, 03:33 PM said:
As for contacting the original person, the problem comes about that my predecessor was unreliable and disorganized when they were actually working this position... they're impossible to find/get information from now.
For references' sake, I ended up taking a 10 uL sample and diluting into 1 mL PBS, taking the OD 600, and sonicating in 15-second bursts at 40% power until the OD dropped by about 60% -- that seems like it will be sufficient for our purposes, as I'm wary of doing it for longer since I obviously don't want to risk denaturing all the assorted proteins we're trying to expose our cells to. Does that seem reasonable?
Check your PM. Found a paper for you.