Principles of cell fractionation and ultracentrifugation as used to separate cell components.

Cell fractionation is splitting cells up into its organelles.

1. The tissue is chopped up and up into a ice cold, isotonic, buffer solution.
2. This is then put in a blender to break open the cells which is called 'homogenisation'.
3. The 'homogenate' is then filtered to get rid of debris like connective tissue.
4. The mixture is spun on a centrifuge, the densest organelle will collect at the bottom.
5. The separated bit at the bottom, the 'pellet' is left in the tube when the homogenate on top which is called the supernatant is poured off into a new tube.
6. This new tube is span again to collect the next densest organelle- this is repeated to collect the desired organelles, with the speed increasing each time.

In step one the liquid is cold to slow down enzymes (that might have been freed from lysosomes) so that they don't digest the organelles. It is isotonic to maintain a normal water potential thereby preventing organelles from bursting with water! Buffer solution maintains the PH so that it is appropriate for the organelles.

There are rules on how fast and long you have to spin the centrifuge to get the desired organelle relating to the order of density. From most dense to least the order of these key organelles goes: nucleus; mitochondria; lysosomes; ribosomes.