Differential centrifugation works by separating cellular components based on their size and density through a series of increasing centrifugal speeds. Initially, the sample is spun at a low speed to pellet larger, heavier components. Then, the supernatant (the liquid above the pellet) is transferred to another tube and spun at a higher speed, to pellet the next largest component, and this process is repeated until all targets are isolated.
Understanding the Basics
Differential centrifugation is a common technique in biology and biochemistry labs to isolate specific cellular components.
- Principle: The core principle relies on the fact that different cell components have different sizes and densities, causing them to sediment at varying rates under centrifugal force.
- Process: The sample, such as homogenized tissue or cells, is placed in a centrifuge tube and spun in a centrifuge. A centrifuge is a device that rapidly rotates samples. The centrifugal force causes the denser particles to move towards the bottom of the tube, forming a pellet, while the lighter particles remain in the liquid, called the supernatant.
Stepwise Process of Differential Centrifugation
The process involves multiple steps with increasing speeds. Here's a typical outline:
| Step | Centrifuge Speed | Components Pelleted | Remaining in Supernatant |
|---|---|---|---|
| 1 | Low Speed (e.g., 1000 x g) | Whole cells, nuclei, large debris | Cytosol, smaller organelles |
| 2 | Medium Speed (e.g., 10,000 x g) | Mitochondria, lysosomes | Cytosol, smaller organelles |
| 3 | High Speed (e.g., 100,000 x g) | Microsomes, small vesicles | Cytosol, ribosomes |
| 4 | Very High Speed (e.g., > 100,000 x g) | Ribosomes | Cytosol |
- Initial Low-Speed Spin: According to the reference, the process begins with a slow speed, which is specifically designed to remove the heaviest particles, such as intact cells, nuclei, and large cellular debris.
- Sequential Increase in Speed: The next step is to increase the speed. This is done to pellet progressively smaller and less dense particles, thereby separating components based on size and density.
- Isolation of Target Components: This step-wise approach allows for the isolation of specific components from the rest of the cell mixture.
Practical Insights
- Choice of Speeds: The exact centrifugation speeds and durations will vary based on the sample and target components.
- Repeated Spins: The supernatant from each spin is carefully removed and transferred to a new tube for the next centrifugation step.
- Fractionation: This approach allows for the separation of cell components based on their density, helping to isolate a fraction that is enriched in the desired components.
Example
Let's say you want to isolate mitochondria from liver cells:
- Homogenize liver tissue to break cells.
- Centrifuge the homogenate at 1000 x g, pelleting nuclei and large debris.
- Transfer supernatant to another tube and centrifuge at 10,000 x g, which pellets the mitochondria.
- Discard the supernatant, and you have a mitochondrial pellet.
Differential centrifugation, as explained in the reference, achieves its separation by a stepwise increase in the centrifugation speed. Lower speeds at the beginning are used to eliminate the heavier food particles from the sample, and the speed is then increased until the targets themselves are pelleted.
