Cell fractionation

• Cell fractionation


  process in which cells are broken up and the different organelles within them are separated out
• Cells are placed in a solution that is:

  • Cold - to reduce enzyme activity that may break down the organelles
  • Of the same water potential (as the tissue) - to prevent bursting or shrinking of organelles due to osmosis
  • Buffered - so that the pH does not change
• Homogenation

  Cells are broken up by a homogeniser (blender) - releases organelles from the cell.

  Homogenate (resultant fluid) filtered to remove large pieces of debris.
• Ultracentrifugation

  process by which fragments in filtered homogenate are separated in centrifuge.

  1. Tube of filtrate is placed in centrifuge and spun at slow speeds.

  Heaviest organelles (nuclei) form thin sediment or pellets at bottom of the tube.

  1. Supernatant (fluid at the top) is removed, leaving only the sediment.
  2. Supernatant transferred to another tube and spun at a faster speed.

  The next heaviest organelles (mitochondria) are forced to bottom of tube.

  • At each increase in speed, the next heaviest organelle is separated out