Recently, I'm trying to lyse the bacterial pellet with sonication to get the protein. Before running the sonication, the pellet was re-suspended into 0,1M PBS buffer. After running the sonication, the suspension become foamed a lot. Is this foaming show that the protein has been denaturated ? and what is the charactericstic if the cell has been lysed ? Thanks
Yes. You can use even water but by the use of detergent makes sure that bacteria was lysed. When you see a foamed sonication is because the sonication tip is in the surface of the mix, you should introduce more the sonication tip into the mix. The foam don´t affect your protein but maybe you do not lyses the bacteria. A key hallmark of lysed bacteria is that the pellet become clear.
Good luck
I would strongly suggest you to add detergent in your sonication buffer to make sure your bacteria are lyzed (use non-ionic detergent like NP40 or Triton X-100, usually 0.05-1%). I agree with the first answer that you should introduce your sonicator tip into your suspension. Foaming is pretty common when you agitate a solution/suspension containing protein. Although it does not usually affect the quality or quantity of your prep, it should be avoided as much as possible. I am sure you already do it, but sonication should be be done on ice in a 4 degree chamber (or cold room). After lysis, your suspension should become more clear than the cell suspension you were sonicating. Also, in my experience, the color becomes slightly darker than the original suspension. Another hallmark of cell lysis is that after centrifugation of the crude lysate, the pellet should be smaller than your original pellet (that you resuspended in your sonication buffer); you might also see some black debris in the middle of the pellet - that's also another sign of successful lysis.
Hi there,
If the cell lysate has foamed it means your sonication condition is too harsh.Try to cool down the sample during the process (the sample should be in a melting ice bath) and to adjust the sonicator settings (power and duty cycle) so that no foam forms. An other line to consider is how deep is the probe into the cell suspension (the probe tip should be right in the middle of the sample avoiding contact with the sample container surface).
I totally agree with DOMINIQUE you have to reconsider your sonication setup and condition this has nothing to do with the PBS buffer good luck
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