You must check that the tip you are using is appropriate for the volume of the sample and for the task you are doing. Why do you know your cells aren't being sonicated correctly? What parameters do you evaluate? If you are checking protein content, maybe your expression system is failing.

PS, Additionally I think I remember that PMSF isn't that soluble in water over 2 mM, so you may want to check that.

Usually you should get along with the lysis buffer you mention and also the conditions you use. As an alternative I can recommend the B-PER Protein Extraction Reagent (Pierce, Thermo Scientific) which works excellent ! Here is a link to the product: http://www.piercenet.com/browse.cfm?fldID=06010401

Good luck !

Thanks for your suggestions...Stephan Schott,Matthew Lalonde,Michael Mühle....

Mr.Stephan Schott,Iam not getting full sonication as i have observed the full turbidity of suspended pellet ever after one 1hr of sonication ,and I have used 1mM PMSF only,accidentally I kept 10mM in my question,sorry for that.

Mr.Matthew Lalonde,I need a detailed explanation about repeated freezing and thawing as how you are doing in your lab.....

- Too long sonication can unfold your protein (1 hour seems to be really a lot, but depends of the strength of your sonicator), which can explain that your protein pellets when you make the centrifugation (I saw your other question)... Putting some ice around your tube during sonication can lower the unfolding problem, but the first thing is to ask people in your lab which conditions they use on the same sonicator to brake their cells.

- Some proteins really don't like sonication, so Matthew's comment is good, as freeze/thaw cycles can perfectly do the job. Just use the same buffer (perhaps up to 10% glycerol could cryoprotect your sample better), freeze with liquid nitrogen, thaw at room temperature by putting your tube in water while stirring, and repeat this twice. This protocol works fine for me.

- Your protein can stick to insoluble material, explaining the loss during centrifugation. Triton and glycerol should reduce this if the interaction is hydrophobic, but not if it is ionic. To get reed of ionic interactions, you can increase the ionic strength of your buffer : usually 0.5M NaCl is good, but sometimes needs to be increased up to 1M if the interaction is really strong (I had the problem with a high affinity DNA-binding protein).

- Eventually, are you sure that your protein is properly folded ? Because, for example, production of an eucaryotic protein in bacteria can give unfolded protein, and this would explain your precipitation problem...

Thanks for your explanation Mr.Yann-Vaï Le Bihan...

I will follow your suggestions......

If indeed your sample isn't sonicating correctly and this isn't just a problem of insoluble protein (i.e. it is in inclusion bodies), I have a few additional suggestions:

a) What size is your sample - if it is larger than say 20-30 ml, then you can use the larger sonicator probe rather than the microprobes. This will enable you to increase the amplitude you are using beyond 50%, which should enable more rapid and efficient lysis. However, you will have to be careful with the increased heating effects - certainly have your sample on ice.

b) Check that the sonicator is set up correctly. Double check the probe is fitted tightly and flush with the sonicator horn to get the system working correctly. Another thing to check for is whether the probe is pitted at all - i.e. has small holes in it - which occurs after extended use. A pitted probe will not work at maximum efficiency. In this case you can either purchase a new probe, or file the current one down to get a fresh, flat surface.

c) Do you see cavitation bubbles in the sample when the sonicator is pulsing - this is a sure sign that things are working correctly.

Having said all this, I too am not a fan of sonication due to the problems of heating and uncertainty over efficiency of solubilisation. Freeze-thaw is a good alternative. I prefer mechanical disruption e.g. with a cell disruptor or homogeniser, although I realise this option isn't always available.

Another convenient method for breaking the cells is with a French press, if there is one available. It takes very little time and doesn't heat the sample (it should be kept in a cold room).

hay santosh i totally agree wid Stephan Schott, how did u come to know r how can u say that ur cell r not sonicated. some times expression system doesnt work resulting no over expressed protein. but r u getting total protein if s means cell r sonicated n expression sytem is not worrking n subsequently if u r not getting total protein then there must be some problem wid sonication n lysis buffer..

Dear Santosh,

Please check the volume and cell mass. If you lyse 100ml culture pellet in 5-6 ml lysis buffer and sonicate at 24-25% amplitude for 5 min ON time, You surely get the lysis.

Freeze-thaw purpose is to do cell rupture by using ice as a knife therefore addition of glycerol will decrease effectivness of this step.

I prefer to do cell rupture with microfludizer (and homogenizer or french press is the same technique), the french press works typically for small volume of a sample.

Dear Santhosh,

The composition of lysis buffer is ok. But I doubt that your cell density may be high. Try to sonicate is diluted condition means for your 100 ml bacterial pellet, suspend it in 6-7.5 ml lysis buffer and keep it on Ice for 30-45 min.( Lysozyme do its activity of bacterial cell wall lysis) Then sonicate at 24-25% amplitude for 20 sec ON and 25 sec OFF cycle on ice for total 5-7 min ON condition. You get better lysis and your protein is also (If Soluble) in native condition. All the Best.

Does sonication for a longer period of time (without generating much heat though) prove to be harmful for the protein in any way?

Yes longer period of sonication may denature your protein. Some protein are very sensitive to sonication, due to local heating. In these cases, French press is more relevant.

How do you know that the lysis is complete?

Lysis can never be 100% in any case in experimental condition but its maximum. you can visualize microscopically, if you want.

You can confirm cell lysis by two ways: 

1. The suspension turns transparent. The turbidity goes down.  If you don't add sufficient DNaseI it also turns dense due to the release of genomic DNA. 

2. You can observe the sonicated cell suspension under microscope and guess the % of cell lysis. 

Another factor that affects cell lysis due to sonication also could be the stage of cell. Stationery phase cells need harsher conditions than log phase cells.