Cell fractionaction and centrifugation

Much of the understanding of cell structure has resulted from scientists’ ability to isolate organelles so that they can be studied. In this process, the tissue being studied is first homogenised (broken up) in a blender to rupture the cell membranes; this is done in an ice-cold, isotonic buffer: 

• Ice-cold to reduce enzyme activity and prevent the organelles being destroyed

• Isotonic to prevent organelles bursting or shrinking as a result of the movement of water in or out of them

• Buffered to ensure proteins (and therefore organelles) are not damaged due to changing pH. 

The liquid is then filtered to remove any large pieces of material, such as tough pieces of tissue that were not properly homogenised. 

Centrifugation

A centrifuge is a bit like a washing machine lying on its side and set on a spin cycle. A centrifuge effectively acts to increase the force of gravity; tubes containing a liquid suspension are placed in the centrifuge and, when turned on, they spin at high speed resulting in any solid material being forced to the bottom of the tube. 

The filtrate from cell fractionation is first run at low speed in the centrifuges, causing the largest (and therefore the most dense) organelles, such as the nuclei, to collect at the bottom of the tube in a pellet. This can then be recovered and studied. The remaining liquid (supernatant) is centrifuged at a higher speed and slightly smaller (and therefore less dense) organelles, such as the chloroplasts, collect in a pellet to be studied. This process continues at increasingly higher speeds to collect less and less dense organelles.

A sample after centrifugation

Definitions