Biology (UP) - Cell Fractionation and Ultracentrifugation

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What is cell fractionation?

  • The process of breaking up cells so their different organelles can be separated.
  • It helps us to study the structure and function of cells and their organelles
  • It is a two step process of homgenation and ultracentrifugation
  • Tissue must be placed in a cold, isotonic, buffered solution
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Why must the tissue be placed in a cold, isotonic,

  • Cold - stops enzyme activity, which may break down organelles
  • Isotonic - prevents organelles bursting/shrinking due to osmotic gain/loss of water. The isotonic solution has the same water potential as original tissue
  • Buffered - keeps the pH constant
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What are the steps for cell fractionation?

1.  Tissue is cut into pieces and placed into a cold, isotonic and buffered solution

2.  Homogenisation: Tissue is homogenised, for example, in a blender or homogeniser. This releases the organelles from the cell because the plasma membrane is broken down and organelles are released into solution.

3. Filtration: Homogenised suspension (homogenate) is filtered to remove any remaining whole cells or large debris e.g. cell wall or membrane. Organelles are smaller than debris, so pass through

4. Ultracentrifugation: The filtrate is placed in a small tube e.g. test tube, and is centrifuged at a low speed. The higher the speed of the spin, the greater the force.

5. The larger fragments collect at the bottom of the tube in a pellet. Smaller fragments remain suspended in a liquid called the supernatant

6. Supernatant is decanted and re-centrifuged at higher speeds for more force until the desired organelle separates into a pellet

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Order of sedimentation as centrifuge speed increas

Pellet 1: Nucleus. Speed of centrifugation x gravity = 1000 with a spin of ten minutes

Pellet 2: Chlorplasts and mitochondria. Speed of centrifugation x gravity = 3500 with spin of ten minutes

Pellet 3: Endoplasmic reticulum, lysosomes. Speed of centrifugation x gravity = 16500 with spin of twenty minutes

Pellet 4: Ribosomes. Speed of centrifugation x gravity = 100000 with a spin of sixty minutes

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What difficulties do scientists need to overcome w

  • Organelles may be too small
  • They might be inside the cell, and so hard to extract
  • Once removed, the cell or organelles may not work
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