Hi there,

Sonication is definitely having effect on protein stability. The problem is that there is no general settings you may rely on. The optimal conditions to preserve protein integrity include a lot of parameters like the size and viscosity of the sample, the temperature of the sample (the colder the better), the settings of the machine (avoid the foam formation in the sample), the probe dimension, the sonicator settings, number of cycles aso... As a summary, it is a very empiric technique. For instance in our lab and for E coli cells (culture of 800mL at OD 1.5-2), cells are resuspended in 40mL of lysis buffer and submitted to 4-5 cycles of 30s  sonication on melting ice with intervals for cooling (Branson Sonifier 250 with large probe; settings: duty cycle 90; output 30-40%).

What is your expression system E. coli? Keep your sample as cold as possible, even stop between sonications to cool it down. As stated there is no recipe, the best thing is for you to establish your own sonication procedure. For this sonicate and visualize the cells to determine when they are broken. 

Hi David, apart from the great advice offered above, have you tried a regular waterbath sonicator? It is not strong enough to disrupt membranes but it can disrupt insoluble aggregates.

Agree with the above.  When I designed my E.coli sonication protocol years ago I tried various sonicators, sonication cycles, number of cycles, cooling between cycles, etc. before I settled on a protocol.  I had my E coli suspension in an ice/salt bath, and avoided foaming.  

You may wish to consider other methods of cell breakage besides sonication. Multiple freeze-thaw cycles can cause breakage. There are also other physical methods for cell breakage, including bead beater, microfluidizer, and French Press. However, since your protein is so delicate, your best bet is probably detergent lysis. The type of lysis buffer to use will depend on the type of cells you are using.

Hi and thank to all.

This Weekend i tested different times/%amplitude in sonication. And I get the monomer ( The protein didnt form aggregates) in one of them ( the method was :20% Amplitude 1sg ON 1s OFF 1min 50 second total time and then how the lysate isn´t too clear I introduce other sonicate step: Amplitude 20% 10sg ON 10sg OFF the same time). But this morning I want reproduced this experiments with other batch ( the difference between both is this batch the induction was to 30ºC and the other was to 20ºC) and other the Buffer ( 50mM TRIS pH:7,6, 200mM NaCl and 10mM glucose instead of 50mM Hepes pH: 7,6, 200mM NaCl and 25mM saccharose ). In this case I didnt obtained the monomer because the protein return to form aggregates.

any suggestions? I want repeat it with the same buffer and the same T of the induction. 

I believe the sonication affects my protein through the formation of aggregates. I am very grateful If anyone know a mild method for lysis cells. If it is possible through using the sonicator. 

Thank you

The difference in induction temperature between the two batches could have had a significant effect on aggregation. It has often been found that expressing a protein that tends to form insoluble aggregates at a lower temperature is helpful at reducing aggregation. You could consider doing the expression at as low a temperature as 16oC. (This usually involves moving the incubator into the cold room.)

Hi Adam. Yes, I know when you induced to low temperatures the aggregation is less than a high temperatures. But when I did the sonication proves the batch are the same, all samples are induced at 20C, and in 3 sonication condictions  the protein formed aggregates. So I suppossed that The temperature wasnt a strong reason to form this aggregates. But maybe the union of a low temperature and a mild sonication would favor the monomer conformation. I will try at 18C.

Thank you

Dear David Vizarraga

Sonication is the most popular technique for lysing small quantities of cells and also it is the third class of physical disruption commonly used to break open cells. And yet this process can damage extracted proteins due their fragility. Sometimes, the lysis of samples in the lab will take between 15 seconds and 2 minutes.

So I recommend to try method described following these steps:

a-Preparation of the cell suspension:

-Cell pellets must be completely suspended in a buffer solution by homogenizing (choose your buffer solution compatible with following analysis, e.g. specific chromatography method).

-Add lysozymes and/ or other additives, if needed (they must be also compatible with separation/ purification means).

-Mix/ homogenize the solution gently under mild sonication until complete suspension is achieved.

b-Ultrasonic lysis: Place the sample in an ice bath.

-For cell disruption, sonicate the suspension at 60-90 second bursts (using your ultrasonicator’s pulse mode).

b-Separation:

-Centrifuge the lysate (e.g. 10 min. at 10,000 x g; at 4°C). Separate the supernatant from the cell pellet carefully. The supernatant is the total cell lysate.

-After filtration of the supernatant, you obtain a clarified fluid of the soluble cell protein.

Hoping to be helpful

Marius

Yu could try to use lyzosyme instead 

Because it can damage the protein, you have to optimize all the conditions experimentally.

I Would not say that sonication affect protein integrity if your protein of interest is an aggregated/inclusion body. Because the structure is linear at the aggregated stage and you need to anyway refold the protein after you denature-purify. As everyone said try out different conditions. If your protein is tough to solibulise i suggest change the vector.