Dear All,
Sonication is a vital step in protein purification and over sonication can definitely damage the secondary structure of the protein(correct me if I am wrong). What is the best Hz value for sonication? Is Amplitude different from Hz? or both are same? Sorry for asking this stupid question. I have used two types of instruments: one only shows Amplitude value ,other shows both.
The important things to remember when sonicating your sample are to minimize heating of the sample and to avoid foaming. The sample should be set in an ice water bath to keep it cool. Keep the tip well below the surface, about half-way down into the sample. Keep the cycles short, no more than 30 seconds. Let the sample cool for a few minutes after each sonication cycle. It may take several cycles to get thorough cell breakage. When starting a cycle, begin with low amplitude and increase it gradually until you hear the proper sound, which is like something frying in oil and is quite loud (where ear protection), and you should see agitation at the surface. If the amplitude is too high, foaming will occur and you should immediately stop and let the sample rest.
The sonicator can indeed unfold your protein, so you should be very careful with the power output (amplitude) that you use. Anyway the right value is not a standard as it changes from instrument to instrument. Not only, it depends on the probe and the volume that you are using, generally it is a try and error procedure, you should try first on the lysis buffer and check that the temperature of the buffer does not increase much in an ice bath. The best way to set it up is always alternate on/off periods: something like 2 sec on, followed by 8 sec off. I hope this help.
The important things to remember when sonicating your sample are to minimize heating of the sample and to avoid foaming. The sample should be set in an ice water bath to keep it cool. Keep the tip well below the surface, about half-way down into the sample. Keep the cycles short, no more than 30 seconds. Let the sample cool for a few minutes after each sonication cycle. It may take several cycles to get thorough cell breakage. When starting a cycle, begin with low amplitude and increase it gradually until you hear the proper sound, which is like something frying in oil and is quite loud (where ear protection), and you should see agitation at the surface. If the amplitude is too high, foaming will occur and you should immediately stop and let the sample rest.
For me the correct answer would be: The minimum amplitude you need to break the membrane with no overheating your sample. Normally you would be able to find this at every instruction manual of any sonicator. In case you don´t have the manual, just try at 50-70% of the maximum power. This should work in 90% of the sonicators.
It depends on various factors like sample volume, sample viscosity, cell concentration, sonicator wattage, and probe diameter. Ideally, 1/10 culture volume is optimum. Lets say, pellet of 100 ml culture should be dissolved in 8-10 ml of sonication buffer to get ideal viscosity of sample of be sonicated or 4gram cell pellet per 100 ml of sonication buffer. Too much cells make solution viscous due to genomic DNA and will result in incomplete lysis of cells.
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