Buoyant density centrifugation

Buoyant density centrifugation is a specialized technique used in biochemistry and molecular biology for the separation of biomolecules and cellular components based on their density. This method involves the use of a density gradient, typically created by a concentrated solution of a substance such as sucrose, cesium chloride, or iodixanol, in which samples are centrifuged at high speeds. The principle behind buoyant density centrifugation is that particles will migrate until they reach a point in the gradient where their density is equal to the surrounding medium, thus becoming "buoyant" and forming distinct bands at specific densities.

Principle

The principle of buoyant density centrifugation relies on the differential migration of particles in a centrifugal field through a medium with a gradient of density. When subjected to centrifugation, particles move until they reach a region where their density matches that of the surrounding gradient medium. At this point, they experience no net force and thus stop migrating, effectively separating based on density. This method is particularly useful for isolating nucleic acids, viruses, organelles, and other cellular components with distinct densities.

Applications

Buoyant density centrifugation has a wide range of applications in the fields of molecular biology, genetics, and biochemistry. It is commonly used for:

• Isolation of DNA and RNA from cells and viruses
• Separation of lipoprotein fractions in blood
• Purification of viruses and bacteriophages
• Isolation of cell organelles such as mitochondria, chloroplasts, and nuclei
• Analysis of cell cycle stages by separating cells of different densities

Procedure

The procedure for buoyant density centrifugation involves several key steps: 1. Preparation of the density gradient medium, which is carefully layered into a centrifuge tube. 2. Sample preparation and layering onto the top of the gradient or mixing with the gradient medium. 3. Centrifugation at high speeds, which causes particles to migrate through the gradient until they reach their buoyant density. 4. Collection of separated components, often involving careful fractionation of the gradient to isolate specific bands.

Advantages and Limitations

Buoyant density centrifugation offers several advantages, including the ability to separate components based on very small differences in density and the potential for high purity and yield of isolated materials. However, it also has limitations, such as the need for specialized equipment, the potential for sample dilution or damage during processing, and the requirement for careful optimization of gradient conditions.

See Also

• Centrifugation
• Density gradient centrifugation
• Isopycnic centrifugation
• Ultracentrifuge