Biology Forums - Study Force

Hello,

I plan to use sonication to break up my Shewanella and E.Coli microbes into their various components. I was wondering if anybody knows of a website where I can see specific instructions on how to do this. The instructions should include the sonification process, amount of power (watts) the machine needs to have, and the centrifuge process to obtain the various cellular components.

Also, my budget for an ultrasonic cell disruptor is $4000. Does anybody know of a good brand with a sound enclosure and temperature control?

Thank you!!

I believe the protocol has to be specific for the device you're using.

For instance, I found the following document - I'm not sure if it will help you or not, since I don't have experience with this technique.

Also, I've included a source for another tutorial below.

Sonication is effective, but it will also break open nuclei and other organelles (if that's what you want).

I would suggest other methods of cell disruption too.

There are at least several different options including:

1. Homogenization (i.e. Dounce homogenizer). Often cells are first swollen in a hypotonic buffer to increase their fragility and make them easier to break open. Use Trypan blue to verify that your cells are indeed broken.

2. Nitrogen cavitation. Have not used myself but apparently very effective.

3. Detergent lysis. RIPA may be too strong, as you said, to preserve enzymatic activity. However more gentle detergents (0.5-1% NP-40 or Triton X-100) might be appropriate and typically should not denature proteins. The presence of detergent may complicate downstream purification steps, though.

Whichever of these you choose, I would then use a low-speed spin (say ~1000 x g for 10' at 4 degrees) to remove unbroken cells and nuclei. Then you can use a high speed spin to pellet membranes and you should have a cytosolic fraction as a starting point for your purification.

Let me know if that helps.