I have gone through sonication process for bacterial cells( treated as wells as control ) to estimate total protein. For that 500ml of log phase cultures harvested, washed and concentrated using 50mM Phosphate buffer at pH.7 For sonication process, 50amp with 6s of impulse length for 10mins were applied. On protein estimation, i can get only
Are we talking about E. coli? Anyway 10 min seems too much. What is the power of the sonicator? Size of the tip? Sample size?
Do you have access to a French Press? If you pass your cells through at 18,000 psi 3 times (carefully), you should get essentially 100% cell breakage, without the risk of cooking your extract by sonication, or of getting poor breakage by inadequate sonication.
Dear Omar sir , I want to collect cell free extract from Enterococcus faecalis & Streptococcus infantarius. Both isolated from human feces. to 2ml. Power is around 50amp with 6S impulse strength for 10mins.
Dear Adam sir,
Thank you for your valuable suggestion. But there's no access for French press in our institute. Can you please give me a suggestion with suitable lysis buffer composition. As per literature survey people got the intracellular target components through suitable lysis method. But for me have to quantify the total protein for studying some downstream applications in these bacterial strains.
If sonication is essential, then it should be done carefully to make sure of complete cell breakage and to avoid overheating. I recommend getting a small stainless steel beaker to hold the sample, because heat transfer is better than for glass or plastic. The beaker containing the cells should be set in a bath of ice+water, not just ice, for better heat transfer. The sonicator tip should be a wide one for better breakage. The sonicator should be set to the highest power setting that does not cause foaming. Approach this setting from a low power because as the sample breaks, you will probably have to lower the power setting a little. The tip should be well immersed but not all the way to the bottom of the container. It should make a sound like something frying. It should not foam. You should see substantial roiling movement of the solution. Stop after 30 seconds and examine the suspension under the microscope (you will probably have to dilute it) to see if intact cells are still present. Keep the sample in the ice bath while doing this. This also gives time for the sample to cool down. Continue like this until you can see very few unbroken cells.
Dear Adam sir,
This is awesome.. Thank you a lot for giving me these elaborate explanation which would make me more concerned on sonication process. You have reasoned all the difficulties that i faced during sonication. Its a great help thank you a lot sir. Can i use lysis buffer with the constituents of DTT & PMSF prior to sonication process which make all the cells to be fragile for sonication...
You can certainly include DTT and PMSF in the lysis buffer. DTT helps keep protein thiols in the reduced form. PMSF is a serine protease inhibitor. I don't think they affect the fragility of the cells.
Do you have access to a microfluidizer? I get my best protein yeild with ours on 100 psi. Sonication and bead beating never worked well for me.
Dear Laura Sir,
No we dont have microfluidizer. We have only sonicator Thank you for your Suggestion sir..
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