Differential Centrifugation: Videos & Practice Problems

Protein purification involves differential centrifugation, a stepwise process that separates cell components based on their sedimentation coefficients (s values). During centrifugation, insoluble proteins form a pellet at the bottom, while soluble proteins remain in the supernatant. By adjusting spinning velocities and times, specific organelles like nuclei and mitochondria can be isolated. Understanding the relationship between molecular weight and sedimentation is crucial, as larger mass does not always correlate with higher s values. This technique is essential for obtaining purified proteins for further analysis.

Differential Centrifugation

So after protein extraction and obtaining our proteins, the next step in our protein purification strategy is to perform differential centrifugation. Now, before we talk about differential centrifugation, let's first talk about centrifugation. Centrifugation is the process that's exactly what our crude extract is that resulted from protein extraction. Our 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 centrifugation. 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 it with our sample. And so our sample is going to contain a mixture of a bunch of different proteins.

And so what's important to know is that insoluble proteins or particles, which are particles that do not dissolve, they form solids or precipitates. And these insoluble proteins that form these precipitates are 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 supernatant. So the supernatant is literally the leftover liquid solution above the pellet that contains the more soluble unprecipitated solutes 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.

Svedberg units (s) characterize the speed of sedimentation.

By sedimentation, all we mean is the settling of these molecules to the bottom of the spinning container to form a pellet. Basically, the idea here is that the greater the s value, the faster the sedimentation and the faster the movement of the particle towards the bottom of the spinning container to form a pellet. 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. Examples would include the densities of both the particle and the solvent, as well as the shape and the mass of the particle.

In our example of centrifugation, what you can see again is that we've got our centrifuge, this big gray instrument, and we've got our sample in the spinning container. Before starting the centrifugation, notice that all of our proteins, which are our red proteins and our dark blue proteins here, they are all suspended protein with a low s value, and the dark blue protein represents proteins that have a high s value or a high sedimentation coefficient.

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 pellet ,referred to as the supernatant, 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, which has our supernatant 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 supernatant liquid into a new container and the pellet stays 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 dark blue proteins with the high s value stuck to the bottom of this other container.

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. Molecular weight in grams per mole for each of these proteins. And what you can see is that the tendency is for molecules that have a larger molecular weight to have a larger s value, but that's not the case in every situation. So if we compare ribonuclease A to cytochrome c, what you'll see is that cytochrome c has a smaller molecular weight than ribonuclease A, but its sedimentation coefficient is actually larger than ribonuclease A's sedimentation coefficient. And so what this is saying is that the mass or the molecular weight of the protein is not the only contributing factor to the s value. Even though ribonuclease A 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, in our next video, we'll be able to get a little bit of practice with these concepts, and then in our next lesson video, we'll talk about differential centrifugation. So, I'll see you guys in that practice video.

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.

Answer: (E)

Differential Centrifugation

So now that we've reviewed the basics of centrifugation, in this video, we're going to focus on differential centrifugation. 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 centrifugation. Differential centrifugation is stepwise centrifugal separation of organelles and other cell components using very precise spinning velocities with a bunch of different test tubes here. Our first test tube has our crude extract, which is the result of our protein extraction. At the bottom of each of these test tubes, we have the pellet contents after centrifuging the samples. Notice that originally, we have no pellet contents 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 spin it for a very precise amount of time at a very precise velocity, then we're able to pellet very particular cell components that have similar sedimentation coefficients or similar s values. We'll see a separation with a pellet at the bottom and a supernatant up above, containing the unpelleted material. The pellet in this first separation contains nuclei. So, all of the nuclei of all the cells are pelleted, whereas all of the other cell components are still in cell suspension in the supernatant. The pellet is stuck to the bottom of the spinning container, but the supernatant is liquid, and all of these components are dissolved in that liquid. If we take our test tube and literally pour its contents into a new test tube, the supernatant can be transferred, but the nuclei, the pellet, will stay stuck to the bottom of that first spinning container.

We can then take our supernatant, pour it into a new test tube, and spin this test tube again in a second step at a different spinning velocity, for a different amount of time, and then we are able to pellet a different cell component. We've done that, we poured over, and this time we pelleted the mitochondria. All of the other cell components remain in the supernatant dissolved in solution. Again, the mitochondria pellet is stuck to the bottom of the container. We can take the supernatant liquid solution up above and pour it into a new one. The mitochondria pellets stay stuck to the bottom. We can subject this new test tube here with the supernatant and spin it again at another precise spinning velocity for another precise amount of time. Notice we get another pellet which contains the ribosomes. We're separating out different cell components in these stepwise centrifugal separation steps, separating out different cell components. We can continuously take the supernatant, transfer it over to new tubes, and continuously spin them at precise spinning velocities to pellet different cell components.

Here we've pelleted the insoluble proteins, and then we can pour the supernatant out into a new test tube so that we only have the soluble proteins. Depending on what component we're interested in, sometimes, you're only interested in the supernatant, but other times, you're interested in the pellet. For instance, suppose you were looking for a protein that was inside of the nuclei. Well, you could spin it at this precise velocity, pour off the supernatant, and now you have a pellet at the bottom of this test tube that contains all of the nuclei. It depends on whether you're going to be focusing on the supernatant or whether you'll be focusing on the pellet.

Now, in this situation, we're focused on the supernatant, and we're interested in the soluble proteins. At this point, we've 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. 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. We'll be able to get a little bit of practice with differential centrifugation in our next practice video, and then we'll continue forward with our protein purification strategy. I'll see you guys in that practice video.

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.
• C. To pellet all unwanted proteins.
• D. To fully purify the target protein of interest.

Answer: (B)

Here’s what students ask on this topic:

What is differential centrifugation and how does it work?

Differential centrifugation is a technique used to separate cellular components based on their sedimentation coefficients (s values). After homogenizing cells to obtain a crude extract, the sample is subjected to a series of centrifugation steps at varying speeds and durations. Each step isolates specific organelles or particles by forming a pellet at the bottom of the test tube, while the remaining components stay in the supernatant. For example, nuclei may pellet at lower speeds, while mitochondria and ribosomes require higher speeds. This stepwise process allows for the isolation of different cellular components for further analysis.

What is the difference between a pellet and a supernatant in centrifugation?

In centrifugation, a pellet is the solid mass that forms at the bottom of the test tube after spinning, consisting of insoluble particles or proteins. The supernatant is the liquid that remains above the pellet, containing soluble proteins and other dissolved components. The separation occurs because the centrifugal force pulls denser, insoluble particles to the bottom, forming the pellet, while less dense, soluble particles stay in the supernatant. This distinction is crucial for isolating specific cellular components during differential centrifugation.

How does the sedimentation coefficient (s value) affect the separation of particles in differential centrifugation?

The sedimentation coefficient (s value) indicates the rate at which a particle sediments under centrifugal force. Particles with higher s values sediment faster and form pellets more quickly than those with lower s values. The s value depends on the particle's size, shape, and density, as well as the density and viscosity of the solvent. During differential centrifugation, adjusting the spinning speed and duration allows for the selective pelleting of particles with specific s values, enabling the stepwise separation of cellular components.

Why is differential centrifugation important in protein purification?

Differential centrifugation is crucial in protein purification because it allows for the stepwise separation of cellular components, enabling the isolation of specific proteins. By adjusting the centrifugation speed and duration, researchers can selectively pellet organelles and other particles, removing unwanted components from the crude extract. This process helps to concentrate the protein of interest and reduce contamination from other cellular materials, facilitating further purification steps and accurate biochemical analysis.

What factors influence the sedimentation coefficient of a particle?

The sedimentation coefficient (s value) of a particle is influenced by several factors, including the particle's size, shape, and density, as well as the density and viscosity of the solvent. Larger and denser particles generally have higher s values and sediment faster. However, the shape of the particle also plays a role; elongated or irregularly shaped particles may sediment more slowly than spherical ones of the same mass. Understanding these factors is essential for optimizing differential centrifugation protocols to achieve effective separation of cellular components.