Cell fractionation

• Homogenisation

  • breaking up the cell by grinding it up in a homogeniser or vibrating it
  • this releases cell organelles into a solution called the homogenate
• The homogenate solution should be

  • ice-cold: to reduce activity of enzymes that break down organelles
  • isotonic: to prevent osmosis, so prevents cytolysis/organelles from bursting
  • buffered: to keep pH constant, so prevents enzymes in the organelles from denaturing
• Filtration

  • the cell homogenate is filtered through a gauze to separate any large cell/tissue debris from the organelles
  • organelles are smaller so they pass through the gauze
• Ultracentrifugation

  • the filtrate is poured into a tube, then put into a centrifuge
  • the centrifuge is spun at a low speed - the heaviest organelles are flung to the bottom, forming a thick sediment (pellet)
  • the rest of the organelles stay suspended in solution (supernatant)
  • the supernatant is drained off and poured into another tube, then spun in the centrifuge at a higher speed
  • this process repeats until all organelles are separated
• Order of mass of organelles:

  • nucleus (heaviest)
  • chloroplasts
  • mitochondria
  • lysosomes
  • endoplasmic reticulum
  • ribosomes (lightest)