Biology - Cell Fractionation and Ultracentrifugation

• 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

• 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

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

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

• 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