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5. Protein Techniques



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in this video, we're gonna talk about our fourth step in our protein purification strategy dialysis. So after salting out, it's likely that our protein solution containing our target protein of interest is gonna have a very high salt concentration, because the salting out process requires us to add so much salt. And so it's important to know that many proteins can actually lose their activity if the concentration of salt is too high and some proteins can even be denatured by a high enough salt concentration. So it's in the interest of the biochemist to get rid of all of this salt to make sure that the target protein of interest is not at risk of D nature ation. And so that's exactly where dialysis comes into play. Because dialysis is a process that removes salts and other small molecules via diffusion through a porous, semi permeable membrane and recalled by semi permeable membrane. What we really mean is a membrane that allows the passage of some molecules but prevents the passage of other molecules. And so the way that dialysis works is that our protein solution, which contains our salts, is sealed into a dialysis bag and the dialysis bag acts as a semi permeable membrane, which has a bunch of pours in it. And so the protein salt solution that sealed inside of our dialysis bag is placed into a low salt solution. And over time, eventually, what happens is our proteins, which are fairly large, are going to be retained inside of the bag so they are not allowed to cross the bag. And that's simply because the proteins physically do not fit through the pores in the dialysis bag. But the the salts and the small molecules are actually able to diffuse out of the bag, and they diffuse out of the bag through the pores and the membrane. And so, really, dialysis is not separating proteins from other proteins. It's separating the mixture of proteins from salts and other small molecules, and so we'll be able to see how dialysis works better down below in our example. And so notice over here on the left side of our example, what we have is at the start of dialysis, we have our dialysis bag right here and inside of our dialysis bag. We have our protein solution so you can see that we have these red molecules and these dark blue molecules and the dark blue molecules are our proteins and noticed that their larger than the red molecules and the red molecules are are symbolizing our salts. And so notice that at the start of dialysis that we have a very high salt concentration inside of our dialysis bag. And that means that our proteins are at risk of potential de nature ation due to this high salt concentration. And so essentially, what you'll notice is that the solvent here has a very low salt concentration at the start of dialysis. So over time, eventually, what happens is these red salts are able to diffuse through the dialysis membrane, so they leave the dialysis membrane into the low salt solution through diffusion. And so over time. Eventually, what you'll get is you'll get all of these salts inside of the solvent and inside of the bag, noticed that we have reduced drastically the concentration of salts. So we've gotten rid of a lot of those salts, but are proteins are still retained inside of the bag, and so then we can take out the bag, and we've gotten rid of a lot of that salt and are proteins are no longer at risk of denatured rations. So over here on the right, what we have is is a different image, essentially of the same process. So notice that this blue structure that's going all the way across is representing our dialysis membrane. So that's representing the actual bag itself that we see over here on the left and notice that the dialysis membrane has all of these different pours in. It has all of these holes, and the holes are actually pretty small and looking over here. What we'll see is that the membrane pores are actually about angstrom in length, according to this diagram, whereas the proteins, which are these big green balls here the proteins are very large, so they are 160 angstrom and length here. And so what you'll notice is that the proteins are way too big to fit through these tiny, tiny pores. So 160 angstrom is much bigger than 24 angstrom, and so the proteins. They basically bounced right off of this dialysis membrane, and they're retained inside of the bag. Now, notice that the small molecules such as these little molecules here and the salts, which have much smaller diameters. They are able to fit through the pores, and they actually diffuse through the dialysis membrane. And this is how the process of dialysis works. It essentially removes are proteins, or it, um, purifies our proteins from small molecules and salts. But it doesn't really separate proteins from other proteins. All of the proteins are retained inside of the bag, so that means that we still need to continue to purify our target protein of interest and our next protein purification technique. And so this concludes our lesson on dialysis, and we'll be able to get a little bit of practice in our next couple of videos, so I'll see you guys there.

Dialysis is a technique used to:


Which of the following is a procedure using membrane bags to separate molecules based on molecular size?