Cell fractionation

• 1. Homogenisation - breaking open of cell to get access to organelles
  2. filtration of homogenate - removes unbroken cells, cell debris and membranes
  3. differential ultracentrifugation - where filtered homogenate is spun at gradually increasing speeds each time to separate different organelles in the mixture by size/density/mass
  4. nuclei - mitochondria/chloroplasts - lysosomes -endoplasmic reticulum-ribosomes
• pellets and supernatent

  • after each spin organelles collected at bottom called pellets with unseparated organelles on top called the supernatant
  • supernatant then poured into new test tube leaving pellet in last one
  • new test tube spun again at faster pace to form a pellet containing the next heaviest organelle
• tissue needs specific conditions

  • cold - reduce enzyme activity to prevent enzymes from destroying organelles
  • isotonic - same water potential so cells don’t take in water water via osmosis causing them to shrink or burst
  • buffered - constant ph to prevent enzymes and proteins from becoming denatured