Rate-zonal centrifugation separates particles based on their size and shape by using a density gradient and centrifugal force.
Understanding Rate-Zonal Centrifugation
In rate-zonal centrifugation, a sample is carefully placed on top of a preformed density gradient, typically within a centrifuge tube. This gradient consists of a solution where the density increases from the top to the bottom. Common gradient materials include sucrose, glycerol, or cesium chloride.
The Process
Here's how the process works step by step:
- Sample Layering: The sample containing the particles you want to separate is carefully layered on top of the density gradient.
- Centrifugation: The tube is then placed in a centrifuge, and centrifugal force is applied.
- Sedimentation: Under the centrifugal force, particles within the sample will begin to move (sediment) through the gradient.
- Separation by Size and Shape: Larger and more compact particles will move faster through the gradient than smaller or less compact particles. This difference in sedimentation rate is due to variations in their sedimentation coefficients.
- Zone Formation: As particles of similar sedimentation coefficients move together, they form distinct "zones" within the gradient.
- Fraction Collection: After a predetermined centrifugation time, the gradient is fractionated, and each fraction is analyzed to identify the separated components.
Key Factors
- Density Gradient: The density gradient prevents convective mixing and stabilizes the separated zones.
- Sedimentation Coefficient: This coefficient is a measure of how fast a particle sediments under centrifugal force, and it depends on the particle’s size, shape, and density. The larger the coefficient, the faster it will move in the gradient.
- Centrifugal Force and Time: The duration and strength of the centrifugal force are critical for the successful separation of particles.
- Layering: According to the reference, the sample to be analyzed is layered on a preformed density gradient.
Example: Separating Proteins
Rate-zonal centrifugation is commonly used to separate different protein complexes or organelles. For example, you might use it to isolate ribosomes or various protein aggregates based on their sizes.
Summary
| Step | Description |
|---|---|
| 1 | Sample is layered onto a preformed density gradient |
| 2 | Centrifugal force is applied |
| 3 | Particles move based on their sedimentation coefficient |
| 4 | Different particles form distinct zones due to differential movement |
| 5 | Gradient is fractionated to isolate separated components |
Rate-zonal centrifugation is a powerful technique to separate and isolate biomolecules, organelles, and other types of particles, and it is based on how fast those components will move through a preformed density gradient under the effect of a centrifugal force.
