5. Protein Techniques
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in this video, we're going to begin our discussion on our third step of our protein purification strategy and that 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 salt affect proteins just in general. And so salts actually affect protein soluble ity. And it turns out that at very low salt concentrations, most proteins form insoluble solids or insoluble protein precipitates just like the one that we see over here on the right, where we've got these three proteins that air 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 dissolves soluble state and the mechanism behind salting in this 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 solid ability and allow them to become mawr dissolved in their solution. Now, with the process of salting out. On the other hand, we had lots and 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 H 20 interactions the solvent interactions leaving very little H 20 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 gonna 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 soluble ity on the Y axis increasing from bottom to top and notice that the curve is changing throughout our graph. So that means that the soluble ITI 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 of the right. And so you can think of this graph in these three different sections and we're gonna start with green section on the left. So notice that at the very far left here its 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 the same insoluble protein precipitate, and so you can see that each of these blue balls and our image here correspond with salt. And so we have very little to no salt, and we know that that's gonna allow the polar charged amino acids on the perimeter of proteins. Thio interact with each other, so we have Asper Tate residue on this protein on the left and a license 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 what 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, ah, higher level of soluble ity on this y axis and so you can see that are dissolved. Proteins up here are essentially, uh, 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 that 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 gonna 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, ah, huge dip, insoluble ity, and because its corresponding with low levels on our y axis over here. Uh, notice that are proteins going toe reform a protein precipitate, and so we can see that at all of the salt that's being added around it. All of the salt competes with 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, uh, problem or this video here is that at low salt concentrations, the proteins will precipitate at very high salt concentrations. The proteins will also precipitate. And so we need 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 used 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
Which statement best explains the basis of salting out?
Presence of some salt ions weakens ionic interactions between proteins, leading to greater protein solubility.
Too few salt ions can deprive proteins of H2O solvent, leading to protein precipitation.
Addition of salt ions strengthens ionic interactions between proteins, leading to greater protein solubility.
Too many salt ions can deprive proteins of H2O solvent, leading to protein precipitation.
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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 centrifuge ation, the third step in our protein purification strategy is to salt out our proteins, and salting out involves the removal of unwanted proteins based on their soluble it ease. And so the idea here is that the soluble ity differs from protein to protein, and we can see that in our example below with these two different soluble ity curves, the black soluble ity curve and the red soluble ity curve, which are different from one another. And so because the soluble it ease 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 soluble ity that biochemist 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 formulas provided here. And that's just because ammonium sulfate has proven to be effective in the process of salting out and during salting out, we know that the proteins are going to reform. These protein precipitates that air insoluble and the protein precipitates they actually changed their sedimentation coefficient or there 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 sentiment faster in a centrifuge. It's gonna pellet to the bottom, are spinning container faster. And so, uh, this increased asset value allows us to remove the protein precipitates via centrifuge Gatien so the protein precipitates can be removed via centrifuge ation, 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 off of their soluble it ease. 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 soluble ity graph where we have soluble ity on the Y axis and salt concentration on the X axis. And as we increase insoluble ity are protein becomes more and more dissolved. But the lower we go on this soluble it e y axis, the protein precipitates begin to form. And so, uh, 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 centrifuge ation. So we have a big mixture of proteins that all have similar sedimentation coefficient. 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 proteins with the red curve, and notice that even though they're sedimentation, coefficient or similar, there soluble ity curves could be different. And so notice that at this particular salt concentration here that the red protein I'm sorry, the black protein is mawr 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 in the soluble it ease of the two 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 two, uh, soluble it ease is what is what biochemist take advantage of in the process of salting out. And so notice that at the salt concentration, the protein precipitates that air formed by the black protein will increase the s value and allow us to sediment the black protein at the bottom of our spending container as a pellet. And that's exactly what we see here are black protein which is forming precipitates its pellet it 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 spending containers. And so if we were interested, if our target protein had similar soluble it ease to these black proteins, then we would take our We would take the Super Nate and get rid of it, and the black protein would remain stuck to the bottom of our two, 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 super Natan up above. And so what we can do is we can take the liquid super nation that which has are red proteins. And we can transfer the super native 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 noticed that the soluble ity of the red protein decreases down to this point over here. So now the red protein is starting to form protein precipitates, and that's going to increase its s value and allow us to pellet the red protein. And so then we could get rid of the Super Nadin 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 soluble it ease, um, to further purifier protein. But again, even if we were interested in this black protein pellet, here are protein is not perfectly purified. All we've done is we've pellet in all the proteins that have similar, um, soluble ity curves and similar soluble it ease. And the same goes with these red proteins. Over here, all we've done is Pelle. It'd proteins that have similar soluble ity curves and similar soluble it ease. And so this concludes our lesson on salting out and in our next video will be able to get a little bit of practice, so I'll see you guys there.
Salting out consists of adding __________ in order to ________________________.
Ammonium sulfate; alter the net charge of proteins.
Ammonium sulfate; alter the solubility of proteins.
Salt; neutralize acid/base reactions of proteins.
Salt; perfectly purify a protein of interest.