Mycobacterial protein extraction... without chemicals! - (Oct/25/2007 )

Hey all,
I'm attempting to extract some protein from Mycobacerium tuberculosis, which, as you may know, has a tough and waxy cell wall which is tough to break. The other issue is that since I'm going to likely be running my samples on a mass spec, I've been advised against using chemical lysis buffers, or basically anything except water.

I have tried a protocol which alternates sonication (10 mins at 37kHZ) and resting on an ice bath (10 minutes) after heat killing the sample for 10 minutes... but this has yielded very little protein. The sonicator I'm using is a bath sonicator, which I've seen has not gotten great reviews on this site for cell disruption.

Does anyone have any suggestions on a better method of cell wall disruption for a tough organism?

Bath sonicator is very weak. You need a probe sonicator for that purpose.

Maybe some freeze and thaw steps prior to that.

Hi,
To my knowledge we don't have a probe sonicator inside the level 3 lab... however, we do use the bath sonicator for DNA extractions from mycobacterial species, and it seems to do the trick?
Regardless, it's not working for my protein extraction. For the freeze-thaw method, using dry ice, should I be adding something like ethanol to it first? It was also suggested to me to poke a hole in the lid of my sample before freezing it.. is that necessary?

Thanks!