Cell fractionation

• What is cell fractionation?


  It is the process of breaking up cells to separate their components or organelles.
• Why is a cold, isotonic buffered solution used in cell fractionation?


  To prevent osmotic gain or loss and maintain the structure of organelles.
• What does isotonic mean in the context of cell fractionation?


  It means the solution has the same water potential as the tissue.
• What happens if the solution is hypertonic during cell fractionation?


  The cell does not fracheate, preventing damage to the organelles.
• How can changes in pH affect cell fractionation?


  Changes in pH can alter the structure of organelles and affect enzyme functioning.
• What are the two stages of cell fractionation?


  1. Homogenation

  • Cells are broken up by a homogeniser.
  • Resulting fluid is called homogenate.

  1. Ultracentrifugation

  • Fragments in the homogenate are separated using a centrifuge.
• What is the purpose of homogenation in cell fractionation?


  To break up cells and create homogenate fluid.
• What is the result of the homogenation process called?


  Homogenate fluid.
• What equipment is used in the ultracentrifugation stage?


  A centrifuge.
• What happens to the heaviest organelles during ultracentrifugation?


  They pellet to the bottom of the tube forming sediment.
• What is the liquid called that remains after the heaviest organelles have settled?

  Supernatant.
• What is done with the supernatant after the first spin in ultracentrifugation?


  It is poured into another tube for further spinning.
• How is the supernatant processed after the first spin?


  It is spun at medium speed and then at a faster speed to separate more organelles.
• Which organelles pellet to the bottom after the supernatant is spun at medium speed?


  Mitochondria.
• What is the process of ultracentrifugation in cell fractionation?


  1. Place homogenate in centrifuge and spin at slow speed.
  2. Heaviest organelles (nuclei) pellet to the bottom.
  3. Pour supernatant into another tube.
  4. Spin supernatant at medium speed, then faster.
  5. Next heaviest organelles (mitochondria) pellet to the bottom.
  6. Repeat for each organelle.