Cell Fractionation and Ultracentrifugation

Study Notes

Cell fractionation is a technique used to separate cellular components for biochemical and structural analysis.
Homogenization disrupts cells to release organelles and molecules, creating a cellular homogenate.
Homogenization methods:
- Physical: Ultrasound or hypotonic solutions
- Chemical: Detergents
- Mechanical: Grinding and crushing
Enzymatic digestion can enhance homogenization for specific cell types:
- Lysozyme: Degrades bacterial walls
- Trypsin: Degrades animal tissue extracellular matrix
- Cellulase: Degrades plant cellulosic walls

Centrifugation separates particles based on size and density under high gravity.
Differential centrifugation: A series of centrifugations at increasing speeds, separating organelles and macromolecules based on their size and density.
- Produces a series of pellets and supernatants with different compositions.
**Density gradient ultracentrifugation (DGUC): ** Separates particles of similar sizes but different densities.
- Utilizes a density gradient (e.g., sucrose gradient) to separate components based on their buoyant density.
Factors affecting centrifugation:
- Centrifuge speed (expressed as rpm/min or g)
- Particle mass
- Density difference between particle and solvent
- Centrifugation duration

This figure illustrates the step-wise separation of cellular components by DUC.
It highlights how increasing centrifugation speeds result in the precipitation of specific organelles in pellets, while the supernatant contains the less dense fractions.
- This sequential process leads to the isolation of different organelles, starting with nuclei, followed by mitochondria, lysosomes, microsomes, and finally ribosomes.

This figure shows how a density gradient is used to separate particles of similar size but different densities.
After centrifugation, the particles migrate to a position in the gradient where their density matches the surrounding medium.
This allows for the isolation of specific organelles that might be difficult to separate with DUC alone.

Ultrasound can be used to disrupt cell membranes and release intracellular components.
In the case of nucleoli purification, ultrasound can help to release nucleoli from the nucleoplasm, facilitating their isolation.
Ultrasound is often used in combination with other methods, such as differential centrifugation, to achieve efficient separation of organelles.