In this video, we're going to begin our discussion on our 3rd step of our protein purification strategy, and that's salting out. Now before we talk about how salting out directly applies to protein purification, let's slow down, back up a little bit, and talk about how salts affect proteins just in general. And so, salts actually affect protein solubility. It turns out that at very low salt concentrations, we've got these three proteins that are clumping up together. Now, we know from our previous lesson videos that when most proteins fold, it's the polar charged amino acids that accumulate onto the perimeter of proteins. And the non-polar hydrophobic amino acids, on the other hand, accumulate into the interior or the core of proteins. And so, the polar charged amino acids on the perimeter of proteins can interact with the polar charged amino acids on the perimeter of other proteins to form strong interactions that lead to the insoluble protein precipitate. Now, we know that there's a process called salting out, but as you guys might have expected, there's also a process called salting in. And so, the process of salting in is where we add some salts to transition proteins from this insoluble protein precipitate into a dissolved soluble state. And so, with the process of salting in, we transition proteins into a dissolved soluble state. And the mechanism behind salting in is quite complex and beyond the scope of this course. But you can pretty much think of it as the salt competing and decreasing the strength of interactions between proteins. And so, if we decrease the strength of the interactions holding proteins together, then we can increase their solubility and allow them to become more dissolved in their solution. Now, with the process of salting out on the other hand, we add lots and lots of salt to transition proteins out of the dissolved soluble state and back into the insoluble protein precipitate. And so, when we add all of the salt, it leads to too much salt that will actually compete with the H2O interactions, the solvent interactions, leaving very little H2O to hydrate the dissolved proteins. So they end up clumping back together and reforming these insoluble protein precipitates. So down below in our example, we're going to further clear up this idea of salting in and salting out. And notice in our image, we have this graph that is central, and this is the most important part of our image. And so, notice that this graph has the salt concentration on the x-axis increasing from left to right, and it has the protein solubility on the y-axis increasing from bottom to top. And notice that the curve is changing throughout our graph. So that means that the solubility is indeed affected by the salt concentration, just like what we said up above. And so, notice that our graph actually has three different sections to it that are color-coded, and each section has its own image. So we have the green section over here on the left corresponding to this image on the left. We've got the blue section in the middle corresponding to the image at the top. And then we've got the pink section on the right corresponding to the image at the right. And so you can think of this graph in these three different sections. And we're going to start with the green section on the left. So notice that at the very far left here, it's, lowest salt concentration. So we can write low over here because it has the lowest salt concentrations. The blue section corresponds with medium levels of salt concentration, and the pink section corresponds with high levels of salt concentration. And so we already know that at very low salt concentrations, most proteins form insoluble protein precipitates just like the one shown here. And so at low salt concentrations, it's no surprise that we have this same insoluble protein precipitate. And so you can see that each of these blue balls in our image here correspond with salts. And so we have very little to no salt and we know that that's going to allow the polar charged amino acids on the perimeter of proteins to interact with each other. So we have aspartate, residue on this protein on the left and a lysine residue on this protein on the right, and they are interacting to form a strong ionic bond, allowing them to form this protein precipitate. Now with the process of salting in, we know that it is a transition, allowing proteins to transition into a dissolved soluble state. And so really, the process of salting in is represented by this arrow here in our graph, this green arrow that transitions proteins from the low concentration to the medium levels of concentration where the proteins are dissolved, and they're dissolved because they have a higher level of solubility on this y-axis. And so you can see that our dissolved proteins up here are essentially surrounded by the appropriate amount of salt ions. And so these salt ions are able to interact with the polar charged amino acids and decrease the strength, weaken the strength of those interactions, allowing for the proteins to become dissolved. Now, with the process of salting out on the other hand, we know that we're going to continue to add more and more salt. We're going to add lots and lots of salt to transition the protein from medium levels of salt into high levels of salt. And so, essentially, we're transitioning from the blue dissolved area into this pink area here. And so notice that our curve takes a huge dip in solubility, and because it's And so we can see And so we can see that at all of the salt that's being added around it, all of the salt competes with the water interactions, leaving very little water to hydrate and dissolve the protein. So they clump back up to reform these insoluble protein precipitates. So down below in this blank here, we can put protein precipitates. And so one of the main takeaways from this, problem or this video here, is that at low salt concentrations, concentrations in order to just the right level of salt concentration in order to get dissolved proteins. And so in our next video, we'll talk about how biochemists use this process of salting out here to further purify a protein of interest. So we'll be able to get some practice with these concepts in our next video, and I'll see you guys there.
Salting Out - Online Tutor, Practice Problems & Exam Prep
Created using AISalting out is a protein purification technique that exploits differences in protein solubility at varying salt concentrations. At low salt levels, proteins precipitate due to strong interactions among polar charged amino acids. As salt is added, proteins transition to a soluble state (salting in) by weakening these interactions. Conversely, excessive salt leads to precipitation (salting out) as water interactions diminish. Ammonium sulfate is commonly used, enhancing sedimentation coefficients for effective separation via centrifugation. This method, while not perfect, significantly reduces unwanted proteins, necessitating further purification steps.
Salting Out
Video transcript
Which statement best explains the basis of salting out?
Salting Out
Video transcript
So now that we understand the fundamental basis of salting out, let's talk about how salting out can be used to further purify our protein of interest. And so after differential centrifugation, the 3rd step in our protein purification strategy is to salt out our proteins, and salting out involves the removal of unwanted proteins based on their solubilities. And so the idea here is that the solubility differs from protein to protein, and we can see that in our example below with these two different solubility curves, the black solubility curve and the red solubility curve, which are different from one another. And so because the solubilities differ from protein to protein, this also means that the salt concentration at which proteins precipitate or salt out also differs from protein to protein. And really, it's this difference in solubility that biochemists take advantage of in the process of salting out. And so during salting out, salt is slowly and carefully added to the protein solution, and the salt of choice is usually ammonium sulfate whose chemical formula is provided here. And that's just because ammonium sulfate has proven to be effective in the process of salting protein precipitates, they actually change their sedimentation coefficient or their s value, and they change it in such a way that it's actually increased. And so the increased s value means that it's going to sediment faster in a centrifuge, it's gonna pellet to the bottom of our spinning container faster. And so, this increased s-value allows us to remove the protein precipitates via centrifugation. So the protein precipitates can be removed via centrifugation. And so we'll be able to see that in our example down below.
Now, it's important to note that salting out does not perfectly purify our target protein of interest, but it can remove a significant amount of unwanted proteins based on their solubilities. And so, we're still going to need to use other protein purification techniques after the salting out process. And so in our example of salting out, what you'll see is that we've got our solubility graph, where we have solubility on the y-axis and salt concentration on the x-axis. And as we increase solubility, begin to form. And so, salt concentration increases from left to right on the x-axis. And so in our test tube over here, what we have are the result of our differential centrifugation. So we have a big mixture of proteins that all have similar sedimentation coefficients. But, what you'll notice is, we've got yellow proteins, green proteins, red proteins, black proteins but we're focusing specifically on the black proteins with the black curve and the red protein with the red curve. And notice that even though their sedimentation coefficients are similar, their solubility curves could be different. And so, notice that at this particular salt concentration here, that the black protein I'm sorry, the black protein is more soluble than the red protein at the same exact salt concentration. But if we continue to add salt so if we add more and more salt and we change the salt concentration from this point on the x-axis to this point on the x-axis, notice that there's a pretty big difference in the pro in the solubilities of the 2 proteins. So the red protein is very, very soluble and dissolved, whereas the black protein is not very soluble. It's forming protein precipitates. And this big difference that we see between these 2, solubilities is what prot is what biochemists take advantage of in the process of salting out. And so notice that at the salt concentration, the protein precipitates that are formed by the black protein will increase the s-value and allow us to sediment the black protein at the bottom of our spinning container as a pellet, and that's exactly what we see here. Our black protein, which is forming precipitates, it's pelleted to the bottom of our spinning container as a protein pellet. And so we can see that our pellets are at the very bottom of our spinning containers. And so if we were interested, if our target protein had similar solubilities to these black proteins, then we would take our we would take the supernatant, get rid of it, and the black protein would remain stuck to the bottom of our tube, and we could continue our experiments with that. But suppose we were interested in the red proteins. And so, notice that the red proteins, they remain dissolved up in the supernatant up above. And so what we can do is we can take the liquid supernatant, which has our red proteins, and we can transfer the supernatant over to a brand new, container. And the black proteins, they remain stuck to the bottom of the first container, so they don't get transferred over. And so, at this point, what we can do is we continue to add more and more salt so that we're changing the salt concentration from this point over here over to this point over here. And at this point, where the salt this salt concentration, notice precipitates, and that's gonna increase its s-value and allow us to pellet the red protein. And so then we could get rid of the supernatant and have our red proteins at the bottom of our container. And so, you can see here how salting out really uses a stepwise process and differences in solubilities, to further purify our protein. But, again, even if we were interested in this black protein pellet here, our protein is not perfectly purified. All we've done is we've pelleted all the proteins that have similar, solubility curves and similar solubilities. And, the same goes with these red proteins over here. All we've done is pelleted, proteins that have similar solubility curves and similar solubilities. And so this concludes our lesson on salting out and in our next video, we'll be able to get a little bit of practice. So I'll see you guys there.
Salting out consists of adding __________ in order to ________________________.
Here’s what students ask on this topic:
What is the principle behind the salting out technique in protein purification?
The principle behind the salting out technique in protein purification is based on the differential solubility of proteins at varying salt concentrations. At low salt levels, proteins tend to precipitate due to strong interactions among polar charged amino acids on their surfaces. As salt is added, these interactions weaken, increasing protein solubility (salting in). However, at high salt concentrations, the salt competes with water molecules for hydration, leading to protein precipitation (salting out). This method exploits the unique solubility profiles of different proteins to separate them effectively.
Created using AIHow does ammonium sulfate facilitate the salting out process?
Ammonium sulfate is commonly used in the salting out process because it is highly effective at precipitating proteins. Its high solubility in water allows for precise control over salt concentration. As ammonium sulfate is added to a protein solution, it competes with proteins for water molecules, reducing the hydration layer around the proteins. This leads to protein aggregation and precipitation. Additionally, ammonium sulfate increases the sedimentation coefficient (s value) of protein precipitates, making them easier to separate via centrifugation.
Created using AIWhat is the difference between salting in and salting out in protein solubility?
Salting in and salting out are two processes that affect protein solubility differently. Salting in occurs at low to moderate salt concentrations, where the added salt ions weaken the interactions between polar charged amino acids on protein surfaces, increasing protein solubility. Conversely, salting out happens at high salt concentrations, where the salt ions compete with proteins for water molecules, reducing the hydration layer around the proteins and causing them to precipitate. These processes are utilized in protein purification to exploit differences in protein solubility.
Created using AIWhy is salting out not sufficient for complete protein purification?
Salting out is not sufficient for complete protein purification because it primarily separates proteins based on their solubility differences at varying salt concentrations. While it can significantly reduce the amount of unwanted proteins, it does not achieve perfect purity. Proteins with similar solubility profiles may still co-precipitate, necessitating additional purification steps such as chromatography or electrophoresis to achieve the desired level of purity. Therefore, salting out is often used as an initial step in a multi-step purification process.
Created using AIHow do biochemists use salting out to purify a specific protein of interest?
Biochemists use salting out to purify a specific protein of interest by exploiting the unique solubility profiles of different proteins. They gradually add a salt, typically ammonium sulfate, to the protein solution. As the salt concentration increases, proteins with lower solubility precipitate first. By carefully controlling the salt concentration, biochemists can selectively precipitate and remove unwanted proteins. The target protein, which remains soluble at a specific salt concentration, can then be isolated. Further salt addition can precipitate the target protein, which is then collected via centrifugation for subsequent purification steps.
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