As you probably have noticed, sonication of E. coli has several risks for our recombinant proteins. This is due to the ultra-sonication procedure, through cavitation, can rise the temperature quickly inside the cell suspension. Therefore short pulses are preferred over a long continuous pulse and it is very important to maintain the cell suspension in chilled ice during sonication. Otherwise, sometimes aggregation can be induced by this procedure in your target protein triggering to insolubility.
Maybe there is not a perfect sonication procedure which can be used for all the recombinant proteins, possibly it will be depend on the expression levels of the rec-protein, but I think that we can find out a condition range in which all the people working with recombinant protein performed in E. coli could work without too much risks.
I would appreciate very much if you share your knowledge in this controversial issue, sharing your sonication conditions. Sometimes in biblio, conditions are not well defined, it is necessary to know: type of machine, diameter probe, cell suspension volume, vibration amplitude and % used, time pulse on/off and the whole time procedure. In my case:
SonoPlus, 3 mm diameter probe (with 210 um amplitude capacity), 20% vibration amplitude, 5´´ on/5´´ off for 30´´ (x3) with 10´´ rest between 30´´ cycles, 5-10 ml cell suspension.
Any other hints will be welcome!
I think it is important to standardize sonication condition for consistency in the protein yeild, purity and monodispersity. I have no clear evidence for this statement, but my experience say so. As yo rightly pointed out there are no good refernces providing the possible problems one could face for not having followed an optimal sonication conditions. Scaling up issue in sonicator has also been challenging. I use 6 cycles of 20 sec ON/ OFF with 50-60% amplitude and 50% duty cycle on ice for 3 mm probe. But scaling these conditions to a 9 mm probe did not give similar cell lysis. Could you discuss about how sonication steps are scaled up with consistency ??. Normally I have seen my colleagues using French press for higher cell mass. Can sonication with bigger probe (diameter) replace this ??.. Does the output voltage displayed in the machine always speaks for the consitency ?
We used a 3mm diameter probe for sonicating E. coli cells. the method is as follows: 60-80% amplitude, 60% cycle duty (0.6 s pulse rate) for 20-30 min. We didn't have problem with isolation of the recombinant protein. However, in some cases the recombinant protein was expressed in the insoluble fraction. We also induce the cells at lower temperature (16 degrees) so that the protein is expressed in the soluble fraction.
Thank you Rajeev,
Yes, we have noticed also that a high porcentage of proteins appear in the insoluble fraction and maybe we are promoting this fact at the sonication step.
Your conditions are much stronger than mine....
try using a microfluidizer. it lyses cells very well at 18000 PSI in the chamber.
I do sonication for long peroids in short intervals of 20secs off and 30secs on , to get maximum ouput, I always use ice to protect the sample from proteases and protein temperature. Adding protease inhibitors also helps alot. Till now I havent got problem.
I think it is important to standardize sonication condition for consistency in the protein yeild, purity and monodispersity. I have no clear evidence for this statement, but my experience say so. As yo rightly pointed out there are no good refernces providing the possible problems one could face for not having followed an optimal sonication conditions. Scaling up issue in sonicator has also been challenging. I use 6 cycles of 20 sec ON/ OFF with 50-60% amplitude and 50% duty cycle on ice for 3 mm probe. But scaling these conditions to a 9 mm probe did not give similar cell lysis. Could you discuss about how sonication steps are scaled up with consistency ??. Normally I have seen my colleagues using French press for higher cell mass. Can sonication with bigger probe (diameter) replace this ??.. Does the output voltage displayed in the machine always speaks for the consitency ?
In EMB there are some instructions: https://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/sonication/
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression