I am purifying and enzyme but, I am worried if I use some much energy affect the activity of the enzyme. How much energy from the sonicator, is safe to keep an purified enzyme with high activity, or the enzyme could lose some activity during the sonication?
It's impossible to make a quantitative statement. The best answer is that you should use the smallest amount of energy you can that will break open most of the cells, while avoiding heating the sample up very much. Also, avoid foaming of the sample because proteins can become denatured at the air-water interface. Many short bursts with periods of cooling in between are better than a small number of long bursts. If you notice foaming, turn off the power immediately. Keep the sample immersed in ice water. You can monitor the progress by examining the sample under a microscope to see if the cells are still intact, or use chromogenic spot test as you go along, if one is available for your enzyme.
Dear Diego Hidalgo
as Adam B Shapiro told you it is not simple to define an exact energy value since it depends from the volume of the sample, from the tipe of sonicator tips and from the sonicator power and from the fractionaction of the energy since to avoid increase in the sample temperature, is important to do not perform a single pulse.
in the following link you can find an example of a protocoll that i used in the past perfroming small scale lysis.
https://proteocool.blogspot.com/2021/05/tip-of-week-6-rapid-protein-expression.html
best regards
manuele
While using sonicator, you should be careful about the power, pause and run volumes. I used 30% of power, 5 seconds of pause and 5 seconds of run for 10 minutes. Also my sample was in a box with ice to avoid heating and degradation of my sample. Considering every aspects of your work, you may apply something like that.
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