Hello everyone! This week felt a bit slower, probably because I repeated procedures from the experiments I did last week. Still, I did get to learn one of the next steps in purifying pfu DNA polymerase from the Bl21s, which is called sonication. Sonication is a way of lysing bacteria through the use of sound waves, in other words, we pop open bacteria by blasting sound at them. This is useful in that it makes it much easier to remove the protein from the bacteria, but there is a downside. When we use sonication, the solution heats up from the sound waves, which can be very bad. Basically, heat is the enemy of proteins. Proteins will become unstable when exposed to high temperatures causing the protein to miss fold in a process known as denaturing. However, pfu is a very heat stable protein, hence why it is used in PCR, so it is not in that much danger of becoming denatured. I even ran a SDS-PAGE on the sonicated samples of bacteria, and surprisingly, the more we sonicated the samples, the more pfu we obtained. This is likely because more cells are being lysed, which causes more pfu to be released in the solution.
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| The right most column is a marker, the one left of that is not sonicated and of pretty high concentration. The rest are sonicated but for different periods of time. |
Sonication is more important for what I will be doing next with the bacteria, which is further purifying pfu from it. From what I have researched so far, heat treatment seems like the easiest method, since pfu has such a high heat tolerance, so that is what I will likely do next week. Hopefully, I'll be able to start working on purification and figure out the most effiecient method for that, so stay tuned!
That's great that your bacteria doesn't denature with heat; that opens a lot more possibilities. I never knew about sonication. Can you actually hear the sound? What decibel is it comparable to?
ReplyDeleteIt's so loud! I'd put it around 70 decibels, but what's strange about it is that if you take it out of a solution, it makes no noise. That's probably because it doesn't really have a good medium to project the sound.
DeleteThere are some researchers in my lab who are also running gels like yours, and I know they have to send them to a mass spectrometry lab to get them analyzed. Do you have the equipment and personnel in your lab to analyze it or do you have to send them off, too?
ReplyDeleteNone of the gels I've run so far require the use of a mass spectrometer. All I really do is either shine UV light on it or stain the gel.
DeleteIt seems as if there is a lot of waiting for results in your project, is this true? If so, what do you do to pass the time?
ReplyDeleteI've never heard of sonication, but it sounds very cool. Are you enjoying witnessing/performing all of these different procedures in a real lab? Also, is there a known temperature for when pfu begins to denature?