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What is cell fractionation?
Process where cells are broken up and the different organelles they contain are separated
Why is the tissue placed in a cold, isotonic, buffered solution?
Cold-Reduce enzyme activity that might break down the organelles
Isotonic-Prevent organelles bursting or shrinking as a result of osmotic gain or loss of water
Buffered-Maintain a constant pH to reduce enzyme activity
What is an isotonic solution?
Two solutions, separated by a semipermeable membrane, have equal concentrations of solutes and water
What are the two stages of cell fractionation?
Homogenation
Ultracentrifugation
What is the process of homogenation?
When cells are broken up by a homogeniser (blender)
This releases the organelles from the cell.
Resultant fluid known as the homogenate is then filtered to remove any complete cells and large pieces of debris
What is the process of ultracentrifugation?
Fragments in the filtered homogenate are separated in a machine called ultracentrifuge.
This spins tubes of homogenate at high speeds in order to create a centrifugal force
How does the process of ultracentrifugation take place for animal cells?
1. Tube of homogenate is placed in the ultracentrifuge and spun at a slow speed
2. The heaviest organelles, nuclei, are forced to the bottom of tube where they form a pellet
3. Fluid at top of tube (supernatant) is removed, leaving the pellet of nuclei
4. Supernatant is transferred to another tube and spun in the ultracentrifuge at a faster speed than before
5. Next heaviest organelles, mitochondria, are forced to bottom of tube
6. Process continues
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