In this video, we're going to talk about our 4th 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 going to 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 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 denaturation. 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 semipermeable membrane. And recall by semipermeable 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 semipermeable membrane, which has a bunch of pores in it. And so the protein salt solution that's sealed inside of our dialysis 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 salts and the small molecules are actually able to diffuse out of the bag, and they diffuse out of the bag through the pores in 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 notice that they're larger than the red molecules, and the red molecules 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 denaturation 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, notice that we have reduced drastically the concentration of salt. So we've gotten rid of a lot of those salts, but our 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 our proteins are no longer at risk of denaturation. 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 pores 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 .l Å in length, according to this diagram. Whereas, the proteins, which are these big green balls here, the proteins are very large. So they are a .. Å in length here. And so what you'll notice is that the proteins are way too big to fit through these tiny tiny pores. So .. Å is much bigger than .. Å. And so the proteins, they basically bounce 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 our proteins or it, 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. Bag. So that means that we still need to continue to purify our target protein of interest in 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 - Online Tutor, Practice Problems & Exam Prep
Created using AIDialysis
Video transcript
Dialysis is a technique used to:
Which of the following is a procedure using membrane bags to separate molecules based on molecular size?
Here’s what students ask on this topic:
What is the purpose of dialysis in protein purification?
Dialysis is used in protein purification to remove salts and small molecules from a protein solution. After processes like salting out, the protein solution often contains high concentrations of salts, which can denature proteins or reduce their activity. Dialysis employs a semipermeable membrane that allows small molecules, such as salts, to diffuse out while retaining larger protein molecules. This ensures that the target protein remains intact and functional, making it suitable for further analysis and applications.
Created using AIHow does a dialysis bag work in the process of dialysis?
A dialysis bag works by acting as a semipermeable membrane with pores that allow small molecules to pass through while retaining larger molecules. The protein solution, containing both proteins and salts, is placed inside the dialysis bag. When the bag is submerged in a low-salt solution, the small salt molecules diffuse out through the pores, while the larger protein molecules are retained inside the bag. This process effectively reduces the salt concentration in the protein solution, preventing protein denaturation.
Created using AIWhy is it important to remove salts from a protein solution?
Removing salts from a protein solution is crucial because high salt concentrations can lead to protein denaturation or loss of activity. Salts can disrupt the protein's structure and function, making it unsuitable for further analysis or applications. Dialysis helps to remove these salts, ensuring that the protein remains stable and functional. This step is essential in maintaining the integrity of the protein for subsequent purification and experimental procedures.
Created using AIWhat types of molecules can pass through the semipermeable membrane in dialysis?
In dialysis, the semipermeable membrane allows small molecules, such as salts and other small solutes, to pass through its pores. These pores are typically around 24 angstroms in size. Larger molecules, such as proteins, which are often around 160 angstroms or larger, are too big to pass through these pores and are retained inside the dialysis bag. This selective permeability is what enables dialysis to effectively separate small molecules from larger proteins.
Created using AICan dialysis separate proteins from other proteins?
No, dialysis cannot separate proteins from other proteins. The process is designed to remove small molecules, such as salts, from a protein solution by allowing them to diffuse through a semipermeable membrane. All proteins, regardless of their size, are retained inside the dialysis bag because they are too large to pass through the membrane's pores. Therefore, additional purification steps are required to separate different proteins from each other.
Created using AI