Cell Fractionation

Cell Fractionation

1. Requirements of the solution?

   • Cold- Reduce enzyme activity that might break down the ogranelles
   • Isotonic- Prevents organelles bursting or shrinking as a result of osmotic gain or loss of water.
     • Has the same water potential as the original tissue.
   • Buffered- Maintain a constant pH.

2. Homogenation

   • Cells are broken up by a homogeniser to release the organelles from the cell.
   • The resultant fluid is known as the homogenate
     • This is filtered to remove and complete cells and large pieces of debris

3. Process where cells are broken up and the different organelles separated out

   • The techniques of cell fractionation and ultracentrifugation enables considerable advances in biological knowledge. They allowed a detailed study of the structure and function of organelles.

4. ULTRACENTRIFUGATION

   • Process in which the homogenate is spun at a high speed to create a centrifugal force.
     • 1) Tube of filtrate is spun at low speed.
       • 2) Heaviest organelles, the nuclei, are forced to the bottom to form a thin sediment pellet.
         • 3) Supernatant is removed, leaving the sediment of nuclei.
           • 4) Supernatant is transferred to another tube, and spun in the centrifuge at a faster speed.
             • ~PROCESS IS REPEATED~

5. Order of separation of organelles (and speed of centrifugation)

   • Nuclei- 1 000
     • Mitochondria- 3 500
       • Lysosomes- 16 500
         • Ribosomes- 100 000

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