Prepping proteins for purification: a practical look at lysis, sonication, ultracentrifugation, etc.
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- Опубликовано: 24 авг 2024
- Prepping proteins for purification - From cell pellet to column time. We’ve talked a lot about how protein chromatography methods let us purify proteins out of a mixture by flowing that mixture through columns filled with little beads called resin which interact differently with the different proteins based on properties like size & charge. But there’s some prep work you have to do in order to get that mixture of proteins! You’ve got to break open the cells containing the proteins (lyse them) & separate the soluble stuff from the membranous gunk. So today, as I started a couple preps I thought I’d show you how it’s done in a practical/technical post (don’t worry - there’s the theory stuff too) covering lysis, ultrasonication, PEI addition, ultracentrifugation, & filtration - to the point where you’re ready to load your columns!
blog with full text: bit.ly/ultraso...
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.
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).
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 that to remove crystals from mounting loops so I can reuse them. You might have something similar to clean jewelry. When using a probe sonicator, you want to “go deep” - but not too deep!
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.
Great video. Thank you for all the effort. Thank you for falling in love with science!
Thank you! I hope I can help others fall in love with science as well. Just doing my part to pay it forward
Good content keep it up
Nice summary!
Thanks!
Do you store this 10% PEI, in the fridge 4C or -20C or RT. Also, do you adjust its pH to 7 with HCl ? And do I need some ammonium sulphate in my sample before adding this PEI ?
We stored it at RT. And our stock was at pH 9.3 but many protocols suggest a pH of 7 or 8. You shouldn't need ammonium sulfate for the PEI, but typically you do have some sort of high salt to aid with the lysis.
Great as usual,
What is the purpose adding KCl prior sonication?
Thanks
Thanks! High salt helps disrupt the cell membranes, creates osmotic stress, and thus aids with the lysis
@@thebumblingbiochemist do you use it with NaCl as well? Sometimes I use 0.5M NaCl w/o KCl..
Thank you .. That was a quick response 💐
I typically use NaCl, but that protein was picky and needed KCl
@@thebumblingbiochemist gotcha..
I always learn new things (tricks) from you.. thank you immensely. 👏☺
Hi, have you lysed mammalian cells with Laemelli buffer?
I have not, sorry