Standard sonication protocol rather cannot cause protein fragmentation- the energy is too low. It shouldn't even cause its denaturation. It can be denaturated when you sonicate it too long and overheat the sample.

If you use nickel chromatrograpy other bands may be simply impurities. It is not very common to obtain pure protein only after one-step Nickel purification.

If these are not impurities but the products of degradation of protein you can be sure it's due to proteolytic degradation. Be sure to add 1mM PMSF (fresh-it decomposes in water very fast, in few hours) or special (EDTA-free) inhibitor mix to your lysing buffer, work on ice and try not to store the lysate for too long (it is better to start purificatiion just after the lysate preparation).

To check for your protein run SDS-PAGE gel. Spin bacteria from 2-3 ml of culture, remove the supernatant, add 50ul of simple phosphate buffer or water and 10ul of 6x loading buffer(with SDS and reducing agentA). Vortex it vigorously and boil the sample. Spin it one more time before loading on the gel, to remove cell fragments. Load 5-20 ul per lane. Also run samples obtained after sonication- supernatant and pellet. This way you will confirm that your protein is expressed and that it is soluble.

Hi Gabriele.

you did not mention what kind of host did you use for expression of your protein ?and what does molecular weight of your protein ? if you use E.coli the suitable sonication protcol is 9.9 sec on and 9.9 sec off for 6 min, specially for tuner E.coli. and also the molecular weight has effect, proteins which have high molecular weight are easier to degraded for many reasons including sonication. and i think, many bands do not mean those bands are fragments of your protein, it could be contaminated from native protein of your host.and you said ( I could barely see a band of the same size of my protein) are you sure form the level of expression of your protein, i mean did you have over-expression of your protein ?

Actually you can try the following sonication protocol: 2s(sonication)/4s(rest) for 15min in total on ice. Hope that will help.

Hi Gabriele.

you have mention the amount of culture pellet you used for sonication..kindly give the amount of culture then i can give some suggestion.

Standard sonication protocol rather cannot cause protein fragmentation- the energy is too low. It shouldn't even cause its denaturation. It can be denaturated when you sonicate it too long and overheat the sample.

If you use nickel chromatrograpy other bands may be simply impurities. It is not very common to obtain pure protein only after one-step Nickel purification.

If these are not impurities but the products of degradation of protein you can be sure it's due to proteolytic degradation. Be sure to add 1mM PMSF (fresh-it decomposes in water very fast, in few hours) or special (EDTA-free) inhibitor mix to your lysing buffer, work on ice and try not to store the lysate for too long (it is better to start purificatiion just after the lysate preparation).

To check for your protein run SDS-PAGE gel. Spin bacteria from 2-3 ml of culture, remove the supernatant, add 50ul of simple phosphate buffer or water and 10ul of 6x loading buffer(with SDS and reducing agentA). Vortex it vigorously and boil the sample. Spin it one more time before loading on the gel, to remove cell fragments. Load 5-20 ul per lane. Also run samples obtained after sonication- supernatant and pellet. This way you will confirm that your protein is expressed and that it is soluble.

My host was E. Coli. My protein as a monomer weights about 52 kDa and as a dimer 104 kDa. Expression of my protein happens to be toxic for E. coli, so my protein expression had to be reduced. I sonicated about 7 grams of pellet. I did work on ice and did affinity chromatography immediately after doing Ni chromatography. Next time I would run the pellet as well.

Thanks for your replies they have been really helpful.

It seems to me, you are oversonicating, which would give you perhaps more proteins released and more fragmentation due to proteolysis because of temperature control problems. In the past, I sonicated 3 times with 30 second bursts, and 4 min on ice between intervals, but I also used cell lytic to enhance the breakage. Maybe you can try sonicating less, and check for breakage under a microscope. I don't think I ever sonicated so much, and depending on your vessel, you may be getting a lot of heating which would accelerate degradation of proteins by proteinases. So I think for a 15 second burst, 3 min on ice is what I would use.

Thanks Marcia for your help. I would try next time to sonicate 5 times with 15 second burst and 3 min on ice.

As you mentioned, the protein is toxic to the host E. coli cell. Have you confirmed if your protein got expressed? I’d suggest checking the expression level and the solubility before start purification. Sonication should not be a big problem in your case. But please watch the temperature during the sonication. The temperature can be controlled by keeping the suspension on ice and using a number of short pulses (5-10 sec) with pauses (10-30 sec). Also adding protease to the suspension can help preventing the proteolytic degradation.

Thanks Rong for your help.

If your most intense band is at the bottom, then it may mean that your protein is modified (post translational modification). If your protein is being degraded, then most likely you would have seen light bands BELLOW the intense band. Try 10 second sonication at 50% power. Incubate for equal amount of time on ice as sonication time

You should add some Protease inhibitor to your lysis buffer.

I would recommend reducing your sonication amplitude to 30%, with 5min on, 5min off in 15sec intervals on ice. Be sure to take aliquots at each step for SDS-PAGE analysis. If you get incomplete lysis, you can boil a small amount of your post-sonication/centrifugation pellet (which should contain intact cells, among other things) to see if your protein is present at full-length.

I would also recommend a variety of protease inhibitors (which can be purchased cheaply at the website p212121.com) to be added to your lysis/resuspension buffer.

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

Gabriela, you may need to reduce the amount of pellet that you are sonicating. I noticed everyone has an opinion of how to sonicate, but in reality sonication is about how much energy you are putting into your bacterial suspension. As I recall (without looking it up) that energy is measured in erg per cm2. Here is the thing, most people don't measure that for their sonicators and well there are different tip sizes and difference in the power of your power unit. I would take a portion of you bacteria and determine the best method (time on power setting etc) to break your cells. The easiest method is to look at them and compare before and after treatment and manually count them. If you do not have a phase contrast microscope handy you may measure the protein released by the samples. This is easy you take your cell suspension and sample it for total protein, then after treating the suspension you centrifuge out unbroken cells and cell walls (10,000 xg for 10 min) and measure how much protein is released. If you do several time points for treatment you protein released will reach a point where there is no more protein release, that will be your best treatment.

If your sonication protocol is really causing degradation and those extra bands, then a control lysate with minimal or no sonication should not contain those bands. Running a fresh gel in which you compare no sonication versus intermediate sonication and your original sonication protocol may inform you as to whether this is really your problem. Just make sure to load the same total amount of protein per lane! And then you'll know whether your problem really lies with sonication.