cell fractionation

• Steps of Cell Fractionation:

  1. Tissue is cut into small pieces
  2. Pieces are placed in an ice-cold, isotonic buffer solution
  3. Pieces of tissue are then ground into fragments (homogenised) in a homogeniser
  4. Liquid homogenate is filtered
  5. Filtrate is then spun in a centrifuge
  6. At lower speeds, larger organelles collect at the bottom of the tube in a pellet
  7. The remaining supernatant is spun at increasing speeds
• Why is the tissue placed in an ice-cold, isotonic buffer solution?

  ice-cold - slows down enzyme activity to prevent the digestion of organelles

  isotonic - prevents osmosis from occurring so cells do not burst/shrink

  buffered - maintains pH so enzymes do not denature
• Why is the tissue homogenised?

  to break open the cells to release the organelles
• pellet - contains the larger and denser organelles
• supernatant - liquid above the pellet containing less dense organelles in suspension
• Order of organelle separation in centrifuge:

  1. nuclei
  2. chloroplasts
  3. mitochondria
  4. RER
  5. cell membrane and SER
  6. ribosomes
• Why is the liquid homogenate filtered?

  to remove large pieces of tissue debris