Cell Fractionation and Ultracentrifugation

Cell Fractionation: Homogenization and Separation

• 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

Ultracentrifugation: Principles and Techniques

• 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

Figure 1: Cell Fractionation by Differential Ultracentrifugation (DUC)

• 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.

Figure 2: Cell Fractionation by Density Gradient Ultracentrifugation (DGUC)

• 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.

The Importance of Ultrasound in Nucleoli Purification

• 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.