Homogenisation

• Cell fractionation


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

  2. Cells must be prepared in a cold, isotonic, buffered solution

  3. Homogenisation - cells are blended in the cold isotonic and buffered solution, then filtered to remove large cell debris

  4. Ultracentrifugation - homogenate is spun at high speeds to separate organelles by density, less dense organelles require higher speeds to separate
• Order of organelle fractionation

  • Nuclei
  • Chloroplasts
  • Mitochondria
  • Lysosomes
  • Endoplasmic reticulum
  • Ribosomes
• Ultracentrifugation

  • The homogenate ( filtered solution) is spun at high speeds in a centrifuge that separates the organelles according to density.
  • Centrifuge spins at high speeds and the centrifugal force causes pellets of most dense organelles to form at the bottom of the tube.
  • Remove supernatant at the top to leave the pellet and place into second tube to centrifuge
  • repeated at faster speeds removing the supernatant leaving behind the pellet (isolated organelle)
  • Less dense the organelle is the higher speed needed to separate the organelle.