Cell fractionation

• To prepare for cell fractionation, all tissues must be placed in a solution that is:

  1.cold- reduces enzyme activity as these enzymes could break down the organelles

  2.isotonic- prevents osmosis which could lead to organelles shrivelling or bursting

  3.buffered- maintains the pH, as it could affect organelle structure or enzyme activity
• Stages of cell fractionation

  1.homogination

  • tissue is cut up and kept in a cold, buffered solution

  • cut up tissue is further broken up in a homogeniser

  2.ultracentrifugation

  •homogenised tissue is spun in an ultracentrifuge tube at a low speed for 10 minutes

  • a supernatant and a sediment are left (nuclei at 1000x gravity)

  • substrate is filtered out and the sediment is spun again in an ultracentrifuge at a medium speed (3500x gravity for mitochondria)

  • substrate is filtered out and supernatant is spun again at a high speed (16500x gravity for smaller organelle)