The Digestive system

Separating Cell Components

Separating Cell Components

Microscopes are a good way in which to observe how a cell is composed. However, in order to find out the function of these components the best way is to isolate each of them. There are a number of ways this can be carried out. Each component or organelle has its own characteristics so once a cell is broken (homogenization) the organelles can be isolated (fractionation).

The most commonly used technique for fractionating is differential centrifugation. This is where the organelles are separated with the use of centrifugal force. Very small particles, like organelles, will not separate in a solution like larger particles due to gravity. However, if the solution is put through a centrifugal force (rotated at a high speed) then the particles are pushed away from the axis of rotation in radial fashion. The force on the particles compared to gravity is known as the Relative Centrifugal Force (RCF).

  • The cut tissue is placed into an ice-cold isotonic buffer. This prevents enzyme reactions and osmosis (so that the organelles don’t burst). The buffer prevents any pH changes from occurring.
  • The tissue is ground in a blender to open the cells.
  • The mixture is then filtered which gets rid of any insoluble tissue, like connective tissue and fat. The end result is cell-free extract.
  • First the filtrate is centrifuged at a low speed (1000 x g for 10 minutes). This pellet nuclei that can be resuspended.
  • The remaining supernatant is then centrifuged at a medium speed (10,000 x g for 30 minutes). This pellets chloroplasts and mitochondria that can be resuspended.
  • The supernatant is then centrifuged at high speed (1000,000 x g for 1 hour). This pellets golgo, ER and other membrane fragments that can be resuspended.
  • The supernatant is then centrifuged at a very high speed (3000,000 x g for 3 hours). This pellets ribosomes that can be resuspended.
  • The remaining supernatant is now cytoplasm, free of any organelles.