Sonicating in style! (actually ULTRA-SONICATING in ultra-style). Too energetic to hear, but just right to DNA-SHEAR! Just like there’s ultra-violet light that’s too high frequency (same as saying too high energy or short wavelength) for us too see, there’s ultra-sonic sound that’s too high frequency for us to hear. And even though the waves generated by the probe are ultrasonic, so above our hearing range, the sounds produced by the bursting bubbles as they fragment the DNA to help us rescue our protein definitely are not. Hence the beautiful ear-protectors. 

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) 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. Today I’m teaching a new grad student how to do it, so I thought I’d teach you too! (I’m going through the whole purification with her, but I’m just going to cover the sonication part in today’s post – you can find details about all the other parts in other posts).

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 it’s 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 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.

If you go back to the gloved hand analogy, we’ve been considering the gloved hand together, but the glove doesn’t need to be constrained by the constraints of your arm. If you take the glove off, the arm and the glove can move independently (assuming you have some electrically-powered glove or something – point is, molecules can get increased entropy by breaking into smaller molecules. If you give them enough energy.

This is a probe sonicator – you actually put the wave-generating tip in the sample. we also have a bath sonicator – I use this to remove crystals from mounting loops so I can reuse them. You might have something similar to clean jewelry.

Note: our solicitor is a lot more intense than the type of ultrasound you might be familiar with. For example, in medical ultrasound, waves are sent into the body and a machine records them getting reflected back to see what’s where. And whales use to do something similar. But in our case, we’re sticking the generator directly into the sample. And blasting the power. So don’t worry about this happening in your medical procedure. Ultrasound is considered really safe and is definitely safer than x-rays. 

When using a probe sonicator, you want to “go deep” – but not too deep! It took me a while to get the feel for the right depth. If you’re not submerged enough you’ll get foaming and a high screeching. Early in my PhD-ing, after accidentally making a latte foam, I over-compensated and put it too deep and blew out the bottom of the beaker… Another problem with being too deep is that it interferes with circulation. Also, just like in Harry Potter when they say the staircases like to change, since water’s weird in that its ice is bigger than its water, when the ice under the beaker melts the beaker will sink. So instead of just ice, you want to pack the beaker in snug in a ice + some water mixture. One of my co-workers cut the lid of a styrofoam box to help hold the beaker still too. When he left the lab he gave it to me as a parting gift I treasure!

That ice melts because cavitation generates intense heat, which you don’t want to hurt your protein. So you do it in pulses. Bacteria need a pretty good beating, but insect cells are more fragile and, for them I usually use one second on, 4 off – so for 1 minute of total on time it’ll actually take 5 in, so if I’m doing multiple samples back to back I’ll usually leave one going (after making sure all’s good) while I go prepare the one that’s done for the next step, where we take things ultra ultra -> go from ultrasonication to ULTRACENTRIFUGATION, where we spin the cells REALLY FAST (like 35K rpm fast) to separate the membrane bits from the soluble stuff.

Since you’re spinning so fast, you have to use super sturdy ultracentrifuge tubes and balance them carefully (when you’re spinning at 35K rpm you need to be balanced really well! it’s not like when you’re just doing a low pulse spin on one of those minifuges where you just eyeball it loosely). Instead I start with eyeballing, then adjust to within ~0.02g or so. I get them really close and then pipet in the tiny bit that’s left over in the beaker to even them out. 

and FYI, my protein’s not bright yellow. The yellow is from yellow fluorescent protein (YFP) that we co-express with our protein when doing insect cell expression, so that we can tell that the cells are making protein and monitor them so we harvest them before they lyse on their own… You want to make sure they’re in a safe environment first! Which is why, when we harvest them (collect the cells and remove the growth media) we resuspend them in a nice buffer (pH-stabilized salt water) that’s similar to the intracellular liquid and add protease inhibitors so they don’t get chewed up. 

Note: There are also enzymatic methods to degunk the DNA. In undergrad, I used benzonase nuclease. That’s stuck in my head because I really love that name. It’s a genetically-engineered version of an endonuclease (DNA cutter) from the bacteria Serratia marcescens that cleaves DNA & RNA nonspecifically.

more on boiling and boiling point: http://bit.ly/boilingpoints 

more on recombinant protein expression:  http://bit.ly/proteincleaning 

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 

#scicomm #biochemistry #molecularbiology #biology #sciencelife #science #realtimechem

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