Differential Centrifugation - Video Tutorials & Practice Problems
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1
concept
Differential Centrifugation
Video duration:
6m
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so after protein extraction and obtaining are proteins, the next step in our protein purification strategy is to perform differential centrifuge ation. Now, before we talk about differential centrifuge ation, let's first talk about centrifuge ation and centrifuge ation is the process that uses spinning as well as centrifugal forces in order to separate particles inside of a mixed solution. And that's exactly what our crude extract is that resulted from protein extraction are crude extract is a mixed solution of a bunch of different types of structures and molecules. And so what we can do is we can take our crude extract and put it into a test tube. And then we can take our test tube and put it into a centrifuge, which is just a machine that performs centrifuge ation. And so what you can see down below, in our example is this big gray instrument Here is a centrifuge and notice that we have a test tube inside of it with our sample, and so our sample is going to contain a mixture of a bunch of different proteins. And so it's important to know is that insoluble proteins or particles, which are particles that do not dissolve. They form solids or precipitates. And these, uh, insoluble proteins that form these precipitates they're actually pulled down faster to the bottom of the spinning container as it spins. And so what they do is they form a pellet at the bottom of the spinning container. Now, the leftover liquid above the pellet after spinning is known as the super Nadin. So the Super Nadin is literally just the leftover liquid solution above the pellet that contains the mawr soluble UN precipitated salutes and proteins. And so, really, what describes the behavior of these particles in a centrifuge as they're spinning is the sedimentation coefficient, which has units of Svedberg or the S value. And Svedberg is just the name of the last name of the scientists that described the sedimentation coefficient. And so each particle has a sedimentation coefficient and the sedimentation coefficient characterizes the speed of sedimentation. And by sedimentation, all we mean is the settling of these molecules to the bottom of the spinning container to form a pellet. So basically, the idea here is that the greater the S value is, the faster the sedimentation and the faster the movement of the particle towards the bottom of the spinning container toe form a pellet. And so it turns out that the s value for each particle actually depends on the properties of both the particle and the solvent that the particle is dissolved in. And so examples would include the densities of both the particle in the solvent as well as the shape and the mass of the particle. And so, in our example of centrifuge ation, what you can see again is that we've got our centrifuge, this big, great instrument, and we've got our sample and it's in the spinning container and before spinning, actually starting the centrifuge ation notice that all of our proteins, which are are red proteins and are dark blue proteins here they're all suspended and dissolved and the solution up above this solution shown here now the red protein here represents a protein with a low s value. And the dark blue protein represents proteins that have a high s value or high sedimentation coefficient. And so, after we start the centrifuge and we spin, what you can see is the centrifuge has a rapidly rotating rotor that spins our sample super fast in the instrument that creates a centrifugal force that pulls and separates our sample the components in our mixture. And so what you'll see is that the components the proteins that have a high s value, are pulled to the bottom. They sediment at the bottom of our spinning container, and they form a pellet at the bottom of our container, whereas the liquid that's above so this liquid that is above the the the pellet is referred to as the Super Nadin and the Super Natan still has dissolved proteins that have low s values in them. And so what we're able to do is we're able to take this solution here, uh, which has are Super Natan and our pellet. The pellet is really stuck to the bottom of that spinning container. And so what we can do is take our test tube and pour out the Super Natan liquid into a new container and the pellets they stuck to the bottom of the other container. So basically, what we've done is we can separate out the red proteins into a different container and leave these, um, dark blue proteins with the highest value stuck to the bottom of this other container and so over here in this chart, what you can see is that we have a bunch of different types of proteins here, all these different types of proteins and each of these proteins has a unique s value or unique sedimentation coefficient. And over here, what we have is the molecular weight in grams per mole for each of these proteins. And what you can see is that the the tendency is for molecules that have a larger molecular weight have a larger s value. But that's not the case in every situation. So if we compare rebo nuclear A to cytochrome C, which will see is that cytochrome C has a smaller molecular weight than rebo nuclear A. But it's sedimentation. Coefficient is actually larger. Then Revo nucleus is a is a sedimentation coefficient. And so what this is saying is that the mass or the molecular weight of the protein is not the Onley contributing factor to the s value. And really, even though Revo nucleus has a larger molecular weight, maybe its density or its shape reduces its s value a little bit so that cytochrome C actually has a greater s value. And so that's important to keep in mind. And so in our next video, we're gonna be able to get a little bit of practice with these concepts. And then in our next lesson video, we'll talk about differential centrifuge ation. So I'll see you guys in that practice video.
2
Problem
Problem
Which of the following affects the sedimentation of a particle during centrifugation?
A
Mass.
B
Shape.
C
Density of the particle & solvent.
D
a & b.
E
a, b & c.
3
concept
Differential Centrifugation
Video duration:
5m
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So now that we've reviewed the basics of centrifuge ation in this video, we're gonna focus on differential centrifuge ation. And so, after we perform protein extraction by homogenizing the cells and obtaining the crude extract, the second step in our protein purification strategy is to subject the crude extract to differential centrifuge ation and all differential centrifuge ation is is just stepwise centrifugal separation of organelles and other cell components using very precise spinning velocities for a very precise amount of time. And so, in our example, we're going to talk about a general strategy for differential, centrifuge ation and notice. We have a bunch of different test tubes here, but our first test tube has our crude extract, which is the result of our protein extraction. And at the very bottom of each of these test tubes here, what we have are the pellet contents. And so these are the pellet contents after centrifuge ing the samples and so originally noticed that we have no pellet contents, and that's because all of our contents are in suspension in our solution. But if we take our first test tube and we put it into a centrifuge and we, uh spin it for a very precise amount of time at a very precise velocity spinning velocity. Then we're able to pellet or sediment very particular cell components that have similar sedimentation, coefficient or similar s values. And so that's exactly what we'll see here. If we spend our crude extract, what we'll get is ah, separation will get appellate at the bottom and super Natan up above, which has the UNP l it'd material. And so the pellet in this first separation here contains nuclei, and that's exactly what we see here. So we have all of the nuclei of all that sells pellet it, whereas all of the other cell components are still in cell suspension in the super Nadin. And so remember that the pellet is stuck to the bottom of the spinning container. But the super Natan is liquid, and all of these components are dissolved in that liquid. So if we take our test tube and we literally poured into a new test tube, the Super Natan can be transferred. So notice that are super Natan could be transferred. But the nuclei, the pellet is gonna stay stuck to the bottom of that first spinning container And so what we can do is take our super Natan poured into a new test tube and then spin this test tube again in a second step at a different spending velocity for a different amount of time. And then we're able to pellet a different cell component. And so what you'll see here is that we've done that. We poured it over, and this time we pellet id the red component, which is our mitochondria. And so all of the other cell components remain in the super Natan dissolved in solution. And again, the mitochondria pellet is stuck to the bottom of the container. And we could take the super Natan, uh, liquid solution of above and pour it into a new one. And so ah, new test tube. And so the mitochondria pellets, they stuck to the bottom. And so what we can do is subject this new test tube here with the super Natan and spin it again at another precise spinning velocity. For another precise amount of time and notice, we get another pellet which which contains the ribosomes, and so you can see that we're separating out in these stepwise centrifugal separation steps separating out different cell components, and we can continuously take the Super Natan, transferred over to new tubes and continuously spend them at precise spinning velocities to pellet, different cell components. So here we've pelted the insoluble proteins, and then we could pour the Super Natan out into a new test tube so that we on Lee have the soluble proteins. And so depending on what component were interested in, sometimes you're only interested in the super Nate. But other times you're interested in the pellet. So, for instance, suppose you were looking for a protein that was inside of the nuclear Well, you could spend it at this precise velocity pour off the super Dayton. And now you have a pellet at the very bottom of this test to that has all of the nuclei. So you have all of the nuclear, and then you could continue your experiments with that. So it depends on what your experiments are, what you're interested in, what you're gonna be focusing on, whether you're gonna be focusing on the Super Natan or whether you're gonna be focusing on the pellet. Now, in this situation here, we're focused on the Super Nadin and we're interested in this soluble proteins here, and so a to this point, we have gotten rid of a lot of different components that we don't care about, but we still don't have anywhere near a purified protein. So we've gotten rid of a lot of stuff, which is great. But we have to continue through our protein purification strategy to continue to purify the protein of interest. And so we'll be able to get a little bit of practice with differential, centrifuge ation and our next practice video. And then we'll continue forward with our pure protein purification strategy, so I'll see you guys in that practice video.
4
Problem
Problem
What is the main purpose of differential centrifugation?
A
To make the cells dizzy before purifying proteins.
B
To separate out fractions of cell components with similar S values.
