CELL FRACTIONATION

• What is the purpose of cell fractionation?

  To separate and isolate different components of a cell for further study
• What is the first step?

  Homogenisation: sample must be ice cold (to stop lytic enzyme activity), pH buffered (to prevent damage to organelles), isotonic (to prevent osmotic lysis).

  Sample is blended in the homogeniser to break down cell membrane and wall, then filtered to remove large fragments and whole cells.
• What is the second stage?

  Differential centrifugation: sample is first spun at a low speed in the centrifuge, this removes larger, denser organelles (e.g. nucleus)

  Then supernatant is removed to be spun again (leaving the sediment pellet)

  Differential centrifugation is performed until desired organelle has been collected (spin at increasing speeds)
• Most dense to least dense:

  Nuclei,

  mitochondria,

  chloroplasts,

  endoplasmic reticulum,

  Golgi,

  ribosomes
• What kind of force is created by the centrifuge spinning at a high speed?


  Centrifugal force
• CELL FRACTIONATION STAGES