Ultracentrifugation

Question 1

Why are the cells placed in a cold buffered and some water-potential solution

Answer 1

• to reduce enzyme activity that might break down the organelles
  • is of the same water potential as the tissue - to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water
  • buffered - so that the pH does not change. Any change could alter the structure of the organelles or affect the functioning of enzymes.

Question 2

Homogenisation

Answer 2

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

Question 3

Procces of ultracentrifugation

Answer 3

tube of filtrate is placed in the centrifuge and spun at a slow speed.
• The heaviest organelles, the nuclei, are forced 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.
• The supernatant is transferred to another tube and spun in the centrifuge at a faster speed then before

The next heaviest organelles the mitochondria, are foxced to the bottom of the tube.
• The process is continued at each increase in speed, the next heaviest organelle is separated out.