2.2.6 Cell Fractionation & Ultracentrifugation

Stages of Cell Fractionation:

1. Homogenisation:

• Cells are broken apart using a homogeniser (a blender or similar device).
• This forms a liquid called the homogenate, which contains a mixture of cell organelles.
• The homogenate is then filtered to remove debris and unbroken cells.

2. Ultracentrifugation:

• The homogenate is placed in a centrifuge and spun at different speeds to separate the organelles based on their density.

Steps of Ultracentrifugation:

3. Initial Spin:

• The tube is spun at a low speed.
• The heaviest organelles (e.g., nuclei) settle at the bottom of the tube as a pellet.
• The remaining liquid, called the supernatant, is carefully removed.

4. Subsequent Spins:

• The supernatant is transferred to a new tube and spun at a higher speed.
• The next heaviest organelles (e.g., mitochondria) form a pellet at the bottom.
• This process is repeated, increasing the speed each time to separate lighter organelles like lysosomes and ribosomes.

Conditions for Successful Cell Fractionation:

• The cells are suspended in a cold, isotonic, and buffered solution to:
  1. Cold: Reduce enzyme activity and prevent organelle damage.
  2. Isotonic: Maintain the same water potential as the cells to prevent osmotic lysis or shrinkage of organelles.
  3. Buffered: Prevent fluctuations in pH, which could denature proteins and damage organelles.

Order of Organelle Separation (Approximate):

1. Nuclei (heaviest).
2. Mitochondria (and chloroplasts in plant cells).
3. Lysosomes and endoplasmic reticulum.
4. Ribosomes (lightest).

Applications:

• Allows detailed study of organelles, such as examining:
  • Mitochondria for respiration processes.
  • Chloroplasts for photosynthesis.
  • Nuclei for genetic material.