I want to lyze my E. coli for protein purification but I only have an ultrasound bath, not a sonicator with a probe. Does anyone have any experience of this? Any tips or protocols that can help me.
Hi Anette, for optimal lysis, a probe sonicator is probably easiest, so maybe you can ask around and see if another lab has one?
Otherwise, you can carry out lysis through mechanical stress, for example adding glass beads to your sample and vortexing it. This approach could be preferred to sonication in a bath or freeze/thaw, because you avoid heating of your sample (whether or not this is important is dependent on what you want to do with the lysed cells though or whether your protein of interest is sensitive to temperature changes. In addition, you should follow your lysis efficiency at different steps, regardless of what protocol you decide to use, so that you know when to stop (and that you can get away with the minimal amount of sonication/freeze-thaw/bead beating etc). To do this, pay close attention to the color and texture of your sample. Lysed E. coli will actually change slightly in color, from your brown-grey goo to something more greenish. If you did not add DNase/Rnase ahead of time, your sample will become more "slimey". Adding the enzymes at this point should clear up the "sliminess" thus indicating that it really was RNA/DNA that was released from cells.
Also, take little aliquoits before treatment and at certain steps in between and spin them down hard in a tabletop centrifuge. If lysis ocurrs, you shoud start seeing a difference between pellet (e.g.size) and supernatant (e.g. color).
Finally, other lysing possibilities include osmotic shock, and detergents. Again, this will depend on what you actually want to do with the sample, but you could put cells in a very low salt buffer and thus swell them (which also makes them more sensitive to mechanical stress, e.g. by vortexing or vigorous pipetting) or you could add detergent (most labs have triton X-100 which is not too expensive) or a combination of these.
I hope these ideas help some. Good luck!
It's just a suggestion but maybe you can lysate your E.coli by doing freeze/thaw cycles using liquid nitrogen and a warm (30-37°C) water bath. The use of liquid nitrogen allows you to freeze your sample instantly wich is more time saving than using a freezer. Maybe if you know some co-workers taht use liquid nitrogen for their cells-line storage, they could provide you some. Good luck.
Hi Anette, for optimal lysis, a probe sonicator is probably easiest, so maybe you can ask around and see if another lab has one?
Otherwise, you can carry out lysis through mechanical stress, for example adding glass beads to your sample and vortexing it. This approach could be preferred to sonication in a bath or freeze/thaw, because you avoid heating of your sample (whether or not this is important is dependent on what you want to do with the lysed cells though or whether your protein of interest is sensitive to temperature changes. In addition, you should follow your lysis efficiency at different steps, regardless of what protocol you decide to use, so that you know when to stop (and that you can get away with the minimal amount of sonication/freeze-thaw/bead beating etc). To do this, pay close attention to the color and texture of your sample. Lysed E. coli will actually change slightly in color, from your brown-grey goo to something more greenish. If you did not add DNase/Rnase ahead of time, your sample will become more "slimey". Adding the enzymes at this point should clear up the "sliminess" thus indicating that it really was RNA/DNA that was released from cells.
Also, take little aliquoits before treatment and at certain steps in between and spin them down hard in a tabletop centrifuge. If lysis ocurrs, you shoud start seeing a difference between pellet (e.g.size) and supernatant (e.g. color).
Finally, other lysing possibilities include osmotic shock, and detergents. Again, this will depend on what you actually want to do with the sample, but you could put cells in a very low salt buffer and thus swell them (which also makes them more sensitive to mechanical stress, e.g. by vortexing or vigorous pipetting) or you could add detergent (most labs have triton X-100 which is not too expensive) or a combination of these.
I hope these ideas help some. Good luck!
I have always used a sonicator probe and I think that a ultrasound bath would not give an adequate results.
If you want a mechanical methods, there are french press that could be used to lysis your cells.
Ultrasound bath will not work efficiently ... In addition to all advises already given here you may just make your own lysis buffer - put some lysozyme + several detergents (combinations of ionic and nonionic ) , salt , EDTA and keep pH 7-8 ...
For mechanical lysis of large volumes, I prefer the French press, if you can get access to one. You could also try the ultrasound bath, but monitor the OD600 on appropriate dilutions to make sure that your cells do lyse properly. I've had bad experience with sonicator probes : if a bubble gests trapped underneath, it will create a hopeless foam in no time. Otherwise, adding lysozyme + Triton should work too, if your protein can take it. Leave out EDTA if you plan to perform immobilized metal affinity chromatography (for His-tagged proteins)
Ultrasonic baths will NOT lyse your cells. The power output is much much less than a sonicator with probe. However as others have pointed out, you can lyse with detergents or you can also lyse with freeze-thaw cycles.
In this article (https://www.hielscher.com/probe-type-sonication-vs-ultrasonic-bath-an-efficiency-comparison.htm) they suggest that an ultrasonic probe-type device excels an ultrasonic bath by factor of 1000, and that bath sonication is unevenly distributed through the tank (less reproducible results).
However there are also very interesting bath sonicators like this (https://www.diagenode.com/en/p/bioruptor-pico-sonication-device) where the samples (up to 2 ml) can spin at regular speed in the bath tank, and temperature and other parameters are controlled efficiently. This greatly enhances result reproducibility.
I have had the chance to use such a system and although it appeared less powerful than a probe sonicator, it gave very reproducible results.
For small volumes it is probably the easiest way to control sonication cycles and reproducibility (although it takes more time to lyse the cells than a probe sonicator)
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