Hi guys, I am currently optimising the expression of a protein that is found predominantly in the pellet. However, I am unsure how the sonication condition can be adjusted such that I have a larger amount of that protein in the supernatant, which contains about 10% of my protein of interest. May I know if the dry weight matters? By the way, I am using a sonicator that relies on a certain percentage of sonication output. The condition I have been using is 70% output for 10 sec pulse and 10 sec rest. (4times). Not sure if this helps but thanks in advance for any suggestions.
You can simply check the state of your bacterial cells under a microscope to see if they have been sonicated enough. I'm assuming you are using E.coli? If you see little rod shaped cells swimming around you need to sonicate more. Typically people use a range of sonicator outputs (strength depends on the sonicator and how welll it is tuned) and the cycles can also be changed in frequency. Some people pulse, some people run it cosntantly. Ideally, you do just enough to burst the cells open and no more. In many cases your protein may not be completely soluble or even precipitate upon sonication so you need to keep this in mind, sometimes the buffer pH matters, other times the addition of glycerol might help, it might not simply be that the cells are not lysed. I would usually use more sonication in term of output (100% or so up to the max rating for the sonicator tip/horn) and pulse times (eg. constant duty or 90%) for a between 1 to 4 minutes than you state you use, but it depends on so many factors. If I were you I'd sonicate the cells and then check them under a microscope (you'll need a 100x objective, E.coli are small!). If there are still intact rods go back and do some more.
I tried at 30% output for 10 sec pulse and 10 sec rest for 5-7 min. You can try freeze/thaw cycle using liquid nitrogen and water bath (at 30-37 C). I think your protein is forming inclusion bodies which are precipitated with your cell debris. You can try to redissolve your pallet in 8M urea and try to refold your protein. You can also express your protein using different tags e.g. MBP, GST, SUMO etc.
You can simply check the state of your bacterial cells under a microscope to see if they have been sonicated enough. I'm assuming you are using E.coli? If you see little rod shaped cells swimming around you need to sonicate more. Typically people use a range of sonicator outputs (strength depends on the sonicator and how welll it is tuned) and the cycles can also be changed in frequency. Some people pulse, some people run it cosntantly. Ideally, you do just enough to burst the cells open and no more. In many cases your protein may not be completely soluble or even precipitate upon sonication so you need to keep this in mind, sometimes the buffer pH matters, other times the addition of glycerol might help, it might not simply be that the cells are not lysed. I would usually use more sonication in term of output (100% or so up to the max rating for the sonicator tip/horn) and pulse times (eg. constant duty or 90%) for a between 1 to 4 minutes than you state you use, but it depends on so many factors. If I were you I'd sonicate the cells and then check them under a microscope (you'll need a 100x objective, E.coli are small!). If there are still intact rods go back and do some more.
You can run SDS-PAGE gels with samples sonicated with increasing amount of time. Yield should increase (either soluble or insoluble fraction). Do not sonicate more than its needed. Please keep the sample in ice bath all the time. Hope this helps.
see whether the pellet obtained after sonication is black in color, if so then your are over doing it. i generally use 40% power 5 sec on and 6 sec off for 5 min, i works fine for me[and most important always check the level of ice surrounding vessel during sonication]. I would suggest to reduce the power and increase time period, and as Mr. Navdeep has mentioned check whether you protein are forming inclusion bodies.
Hi Shridhar, I do see dark brown speckles when I pelleted those sonicated cells. Will play around with the above mentioned suggestions. Thanks!
Hi... Pls can someone help me with the details procedure/steps on how to sonicate bacterial pelleted cells?
Thank you
Hi, I am just wondering that is the ribosomal protein is settled in the pellet or stay in supernatant after sonication ?
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