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. Note: our sonicator 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 an 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. But before I spin them I do one more thing to really make the DNA stay away (from my protein). The sonicator sheared it up, so now I have a bunch of DNA pieces floating around and I want to “unfloat them” - take them out of solution (get them to precipitate) so that they’ll pellet out, away from my soluble protein. To do this, I add PEI (PolyEthyleneImine). PEI is a cationic polymer (chain of similar, positively-charged, repeating units). PEI’s repeating units are amine (nitrogen-hydrogen) groups with ethyl (-CH₂CH₂-) linkers, and they can branch off of each other tree-like. DNA (being negatively-charged) is attracted to it, so they snuggle up together, forming a nice compact blob called a polyplex which, importantly is charge-neutralized. Water doesn’t like to surround neutral things because water molecules are highly polar - they have partly positive parts (the H’s) and partly negative parts (the Os) so they’d much rather bind to other partially charged or fully charged things. They exclude neutral things (the hydrophobic exclusion effect) so those neutral things precipitate. It’s really cool to watch - I add PEI drop-wise while stirring on a magnetic stir plate and you can see the solution get all cloudy and the stir bar starts having trouble stirring as it gets thicker. Then, when you super-fast-spin them, they sink. (warning: this can also precipitate some negatively-charged proteins so use with caution if you want to purify one of those! I add from a 10% solution to a final concentration of 0.2% but you might need to experiment especially if you’re worried about accidentally precipitating protein!) 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. After the spin is done it’s on to chromatography, where we use columns filled with little beads (resin) that interact with different proteins differently and thus let us separate them. more on that here: bit.ly/proteincleaning more on boiling and boiling point: bit.ly/boilingpoints more on recombinant protein expression: bit.ly/proteincleaning more on lysis: bit.ly/homogenization & ua-cam.com/video/IPfRDirApoM/v-deo.html more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0
So why would you want to break up the DNA in the first place? What field of study is this device used for? I was just searching for a Magnetic Stirrer when one of these popped up and I got curious.
@@thebumblingbiochemist What is the purpose of doing that? ex[lain to someone who isn't in this field of study. Explain so and electronic engineer can understand what you are doing.
We want to study how specific proteins work. In order to do this, we want to be able to study them in their pure form, where we can manipulate conditions. So, we get bacteria to make the protein for us, then we purify the protein out of the bacteria. There's a lot more in the bacteria than just our protein, so we want to get rid of the other stuff. One of the "goopiest" (most viscous), things in bacteria is its DNA. So we want to break it up so it doesn't interfere with our protein purification. That way, we can more easily isolate our protein. Does that help?
To clean probe sonicator tip, which ethanol should I use (either denatured ethanol or 200 proof ACS grade ethanol? I am going to extract proteins from cell lysate and need to clean sonicator tip with ethanol followed by water between use!
Lactose brought me to this video. Lewis M. (2011). A tale of two repressors. Journal of molecular biology, 409(1), 14-27. DOI: 10.1016/j.jmb.2011.02.023
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.
Note: our sonicator 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 an 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.
But before I spin them I do one more thing to really make the DNA stay away (from my protein). The sonicator sheared it up, so now I have a bunch of DNA pieces floating around and I want to “unfloat them” - take them out of solution (get them to precipitate) so that they’ll pellet out, away from my soluble protein. To do this, I add PEI (PolyEthyleneImine). PEI is a cationic polymer (chain of similar, positively-charged, repeating units). PEI’s repeating units are amine (nitrogen-hydrogen) groups with ethyl (-CH₂CH₂-) linkers, and they can branch off of each other tree-like. DNA (being negatively-charged) is attracted to it, so they snuggle up together, forming a nice compact blob called a polyplex which, importantly is charge-neutralized.
Water doesn’t like to surround neutral things because water molecules are highly polar - they have partly positive parts (the H’s) and partly negative parts (the Os) so they’d much rather bind to other partially charged or fully charged things. They exclude neutral things (the hydrophobic exclusion effect) so those neutral things precipitate. It’s really cool to watch - I add PEI drop-wise while stirring on a magnetic stir plate and you can see the solution get all cloudy and the stir bar starts having trouble stirring as it gets thicker. Then, when you super-fast-spin them, they sink. (warning: this can also precipitate some negatively-charged proteins so use with caution if you want to purify one of those! I add from a 10% solution to a final concentration of 0.2% but you might need to experiment especially if you’re worried about accidentally precipitating protein!)
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.
After the spin is done it’s on to chromatography, where we use columns filled with little beads (resin) that interact with different proteins differently and thus let us separate them. more on that here: bit.ly/proteincleaning
more on boiling and boiling point: bit.ly/boilingpoints
more on recombinant protein expression: bit.ly/proteincleaning
more on lysis: bit.ly/homogenization & ua-cam.com/video/IPfRDirApoM/v-deo.html
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0
Talk Nerrdy to me!
great video, very informative!
Thanks! I'm always happy to geek out about science.
So why would you want to break up the DNA in the first place? What field of study is this device used for? I was just searching for a Magnetic Stirrer when one of these popped up and I got curious.
When you want to purify protein, the DNA just gets in the way!
@@thebumblingbiochemist What is the purpose of doing that? ex[lain to someone who isn't in this field of study. Explain so and electronic engineer can understand what you are doing.
We want to study how specific proteins work. In order to do this, we want to be able to study them in their pure form, where we can manipulate conditions. So, we get bacteria to make the protein for us, then we purify the protein out of the bacteria. There's a lot more in the bacteria than just our protein, so we want to get rid of the other stuff. One of the "goopiest" (most viscous), things in bacteria is its DNA. So we want to break it up so it doesn't interfere with our protein purification. That way, we can more easily isolate our protein. Does that help?
Thank you! You're saving me
So glad I could help! Good luck!
To clean probe sonicator tip, which ethanol should I use (either denatured ethanol or 200 proof ACS grade ethanol? I am going to extract proteins from cell lysate and need to clean sonicator tip with ethanol followed by water between use!
Any ethanol should be fine for this
Thanks for sharing!
Hope you found it helpful!
@@thebumblingbiochemist If possible would you please share the US doctoral and postdoctoral application system and how to be successful in it? Thanks!
hope this helps bit.ly/gradpostdocetc
Lactose brought me to this video.
Lewis M. (2011). A tale of two repressors. Journal of molecular biology, 409(1), 14-27. DOI: 10.1016/j.jmb.2011.02.023