Ultrasonication (often just called "sonication") in cell lysis скачать в хорошем качестве

Ultrasonication (often just called "sonication") in cell lysis

Sonicate good times, come on! The ultrasonicator is a “hot” item (but don’t let it get your sample too hot!) It’s in such high demand because our lab does a lot of protein purification and (once you’ve gotten cells to make your protein of interest) the first step in purifying them is breaking them free from their cellular prisons. The sonicator helps us do this by using ultra-sonic sound waves to make and break bubbles which help lyse (break open) the cells and shear (break into pieces) the cellular DNA so it doesn’t goop up and get in the way. Even though the waves generated by the probe are ultrasonic (have wavelengths higher in frequency than our hearing range can detect), the sounds produced by the bursting bubbles definitely are not. blog form (text old, video is new): https://bit.ly/ultrasonicshearing In protein purification, where you’re trying to isolate one protein, a lot of attention is (rightly) paid to removing other proteins. But there’s a big, gunky elephant in the cytoplasmic room - DNA. And ULTRASONICATION is a way to fragment it so it to reduce the viscosity (syruppiness) of the liquid you get when you break cells open (lyse them) - the LYSATE. RECOMBINANT PROTEIN EXPRESSION is where we get cells (often bacteria or in this case insect cells) to make a protein we tell them to by using molecular cloning to stick the gene for the protein into an expression vector. More in other posts. For now, just go with it and believe me that we can get the cells to make the protein. They’ll make it for us, but they won’t purify it for us, so that’s where protein-purifying biochemists like I come in to take the protein out and remove everything else. Unless you’re expressing a secreted protein, which the cells export, the protein you want is inside the cell. So to get to the protein you have to break the cell open - we call this lysis and there are different ways to do this including freeze-thawing, grinding, pressing, adding enzymes (reaction speeder-uppers) etc. Sonication helps with lysis, but its main benefit in this case is shearing the DNA (causing it to break into fragments). This is important because there’s a lot of DNA in cells (especially if you’re using eukaryotic (non-bacterial) cells, which have a bunch of DNA stored in a membrane-bound compartment called the nucleus). Normally that DNA is all wound up but when you lyse the cells you disrupt those membranes too, so the DNA goes spilling out and, outside the confines of the nuclear envelope it takes the opportunity to spread out. Molecules want freedom. This freedom/randomness/disorder is called ENTROPY and the second law of thermodynamics says that nature likes this. When considering entropy, it’s helpful to think about all the ways something can move. Think about an arm with a glove on. If your arm is in a sling, you have very little freedom to move. Take the sling off and you can move more. But if there are lots of other people near you, your movement is still restricted. The less stuff around, the more you can swing around your arm. But you need energy to do this. The reason water (or any liquid) boils is that the individual water molecules get enough energy to break free from the surrounding molecules. The energy needed to do this comes from heat and is measured as “temperature” and the amount needed depends on the pressure. Under lower pressure, it’s easier to break free, which is why water boils at lower temperatures at high elevations, where air pressure’s lower. Unlike light waves, sound waves require a medium to travel (i.e. they need to be able to “shift stuff” so can’t travel through a vacuum). As the wave travels it literally pushes the molecules closer together & farther apart, creating alternating high pressure & low pressure zones. Ultrasonication involves sticking a probe into the lysate that generates waves of energy that travel through the liquid, creating periodic low pressure & high pressure zones in the liquid. As intense ultrasonic waves travel through the lysate, it’s like they’re taking water up a mountain, then in a submarine, then back up the mountain, etc. When there’s low pressure, water molecules seize the chance to break free - but the probe is in the middle, not at the surface where there’s the greatest chance of a true escape to the air. And, even if these bubbles try to rise up, they’re soon hit by the high pressure part of the sonication, which causes them to collapse - this is called GASEOUS CAVITATION - like how cavities are holes in your teeth, the cavities in this case are little bubbles of gas in a liquid caused by changes in pressure. When the bubbles collapse (and there are millions of them), they send out shock waves that generate mechanical force that can literally shear apart the DNA into smaller pieces. This method is also used to break up DNA for things like DNA sequencing where you need smaller fragments. finished in comments