Cell fractionation

• What is cell fractionation?

  A process used to isolate different organelles.

  -It enables individual organelle structures and functions to be studied.

  -Cells are broken open to release the contents and organelles are then separated.

  -They must be prepared in a cold, isotonic and buffered solution.
• Why must the solution be cold?

  To reduce enzyme activity~ when the cell breaks open enzymes are released which could damage the organelle.
• Isotonic?

  The solution must be the same water potential to prevent osmosis as this could cause the organelles to shrivel or burst.
• Buffered?

  The solution has a pH buffer to prevent damage to organelles.
• What are the three steps to cell fractionation?

  Homogenisation

  Filtration

  Ultracentrifugation
• What is homogenisation?

  The breaking up of cells:

  -It can be done by vibrating the cells or by grinding them in a blender.

  -This breaks up the plasma membrane and releases the organelles into the solution.
• What does filtration involve?

  Getting rid of the big bits:

  -The homogenised solution is filtered through a gauze to separate any large cell or tissue debris.

  -Organelles are much smaller than the debris, so they pass through the gauze.
• Ultracentrifugation:

  .The filtered solution is spun at different speeds in a centrifuge

  .Organelles are separated according to their densities
• Differential centrifugation:

  ~The centrifuge spins and the centrifugal forces cause pellets of the densest organelles to form at the bottom.

  ~A centrifuge is first spun at a low speed and then the process is repeated at increasingly faster speeds.

  ~Each time the supernatant (liquid) is removed, leaving behind a pellet of organelles.

  ~The supernatant is then spun again to remove the next pellet of organelles.
• Order of organelles: (according to size and mass)


  Nuclei

  Chloroplast

  Mitochondria

  Lysosomes

  ER

  Ribosomes