Sucrose is used in density gradient centrifugation primarily because it creates a stable and easily controllable density gradient that allows for the separation of biological molecules or particles based on their size and density.
How Sucrose Facilitates Separation
Sucrose offers several key advantages that make it well-suited for density gradient centrifugation:
- Gradient Formation: Sucrose solutions of varying concentrations are carefully layered in a centrifuge tube, creating a continuous or discontinuous density gradient. The highest concentration (and therefore highest density) is at the bottom of the tube, and the lowest concentration (and density) is at the top.
- Particle Separation: When a sample containing a mixture of particles is layered on top of the sucrose gradient and centrifuged, the particles will migrate through the gradient until they reach a point where their density matches the density of the surrounding sucrose solution. At this point, they will stop migrating and form a band. Particles of different densities will band at different locations within the gradient, achieving separation.
- Stability: Sucrose solutions are relatively stable and do not readily denature proteins or nucleic acids, making them compatible with a wide range of biological samples.
- Ease of Use: Sucrose is readily soluble in water, making it easy to prepare solutions of different densities. The density of sucrose solutions can be accurately controlled and measured using a refractometer.
- Low UV Absorbance: Sucrose has relatively low UV absorbance, which allows for the detection of separated molecules by UV absorbance scanning after fractionation.
Example: Separating Ribosomes
The provided reference highlights the use of sucrose density gradient centrifugation to separate mammalian mitochondrial ribosomes from other large oligomeric complexes within the organelle. After treatment with detergents (to release ribosomes from membranes), the sample is layered on a sucrose gradient and centrifuged. The ribosomes, being denser than many other components, will migrate further down the gradient than less dense complexes, allowing for their purification.
Advantages of Sucrose over Other Gradient Materials
While other materials can be used to create density gradients, sucrose is often preferred because of its:
- Cost-effectiveness: Sucrose is relatively inexpensive.
- Availability: Sucrose is readily available in high purity.
- Compatibility: Sucrose is compatible with most biological molecules and downstream analyses.
Summary
Sucrose is a popular choice for density gradient centrifugation due to its ability to create stable, easily controllable density gradients, its compatibility with biological samples, and its ease of use. It allows for effective separation of molecules and particles based on their size and density, as demonstrated by its use in purifying ribosomes.
