cell Fractionation

• Cell fractionation is the process where cells are broken up and the different organelles they contain are separated out.
• Before cell fractionation can begin, the tissue is placed in a cold, buffered solution of the same water potential as the tissue.
• The solution is ice cold to reduce enzyme activity that might break down/ digest the organelles.
• The solution is the same water potential (isotonic) as the tissues to prevent organelles bursting/lysis or shrinking as a result of osmosis.
• The solution is buffered so that the pH does not fluctuate. Any change in pH could alter the structure of the organelles or affect the functioning of enzymes by denaturing proteins.
• Homogenation:

  • Cells are broken up by a homogeniser (blender). This breaks the plasma membrane and releases the organelles from the cell.
  • The resultant fluid, known as homogenate.
  • It is then filtered to remove any complete cells and large pieces of debris. 
• Ultracentrifugation- the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge. This spins tubes of homogenate at very high speed in order to create a centrifugal force (towards the bottom of the tube).
• Ultracentrifugation (1):

  • The tube of filtrate is placed in the centrifuge and spun at a relatively slow speed 
  • The nuclei are the most dense organelles, and so will travel most quickly to the bottom of the tube, where they form a thin sediment or pellet. 
  • The fluid at the top of the tube (supernatant) is removed, leaving just the sediment of nuclei (which is then collected for study). 
• Ultracentrifugation (2):

  • The supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before and for a longer time.  
  • The mitochondria are separated next. (They are less dense than the nuclei but more dense than Endoplasmic Reticulum)  
  • The process is continued in this way so that at each increase in speed, the next dense organelle is sedimented and separated out.
• Order of ultracentrifugation:

  • Nuclei
  • Chloroplasts
  • Mitochondria
  • Endoplasmic Reticulum/ Golgi Apparatus
  • Ribosomes