2-2-6-cell-fractionation-and-ultracentrifugation

Cell fractionation & ultracentrifugation

• Cell fractionation is a process used to isolate specific organelles from cells for closer study (e.g. under an electron microscope or to investigate organelle function)

• The process has three main stages

  • homogenisation

  • filtration

  • ultracentrifugation

Homogenisation

• Cells are broken up using a homogeniser (blender)

• This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate

• Carried out in a cold, isotonic, buffered solution:

  • Cold: slows enzyme activity

  • Isotonic: prevents osmotic damage to organelles

  • Buffered: maintains pH to avoid protein/enzyme denaturation

Filtration

• Homogenate is filtered through a gauze to remove large debris

• Organelles remain in the filtered solution (the filtrate)

Ultracentrifugation

• The filtrate is placed into a tube and the tube is placed in a centrifuge

  • A centrifuge is a machine that separates materials by spinning

• Filtrate is spun in a centrifuge at increasing speeds

• Heaviest organelles form a pellet at the bottom

• The rest of the organelles stay suspended in the solution above the pellet

  • This solution is known as the supernatant

• Supernatant is re-spun at higher speeds to isolate lighter organelles

• Order of separation (heaviest to lightest):

  • Nuclei

  • Chloroplasts (in plants)

  • Mitochondria

  • Lysosomes

  • Endoplasmic reticulum

  • Ribosomes

• This process is repeated at increasing speeds until all the different types of organelle present are separated out