Cell fractionation

• What are the three stages of cell fractionation?

  Homogenisation, filtration, ultracentrifugation
• What forms at the bottom of the tube during centrifugation?

  A pellet
• What is the liquid that remains above the pellet called?

  Supernatant
• What is the order of organelle separation from heaviest to lightest?

  Nuclei, chloroplasts, mitochondria, endoplasmic reticulum, ribosomes
• Which organelle is collected first during centrifugation?

  Nuclei
• Which organelle is the lightest among those separated?

  Ribosomes
• What is the purpose of cell fractionation?

  Break open cells & remove cell debris so organelles can be studied
• Describe homogenisation.

  • Process by which cells are broken open so organelles are free to be separated
  • done using homogeniser (blender)
• Describe and explain the conditions needed for homogenisation.

  • Cold -> reduces enzyme activity preventing organelle digestion
  • Isotonic -> prevents movement of water by osmosis - no bursting / shrivelling of organelles
  • Buffered -> resists pH changes preventing organelle + enzyme damage
• Describe ultracentrifugation.

  • Homogenate solution filtered to remove cell debris
  • solution placed in a centrifuge which spins at a low speed initially
  • then increasingly faster speeds to separate organelles according to their density