Cell fractionation is separating organelles of a cell from the rest in order to study them individually.
Key Words before we begin!
Fractionation-the process by which cells are broken up & organelles separated out.
Ultracentrifugigation-process by which fragments in the filtered homogenate are separated in a machine called an ultracentrifuge.
Supernatant-the fluid left over after ultracentrifugigation (contains all organelles that haven't been separated into the sediment)
Isotonic-same concentration of sugars etc as in the organelles
Buffered-means to maintain a constant pH.
Step 1: Homogenisation
Homogenisation put simply is breaking up the cells.
It can be done in several ways, but all of them do the same thing, they break up the plasma membrane so the organelles are released into a solution.
The solution must be:
- Ice cold: to reduce the activity of enzymes which break down the organelles
- Isotonic: to prevent the organelles shrivelling or bursting due to osmosis
- Buffered: to keep the conditions constant so organelles aren't damaged.
Examiners often like to give details of an experiment, where the conditions were one of the above. They often ask why they are so, so its very useful to learn the reasons behind the conditions.
Step 2: Filtration
Next, the homogenate (homogenised cell solution) is filtered to separate any cell or tissue debris.
The organelles are much smaller than the debris so the pass straight through into the solution.
TOP TIP 2!
This process doesn't separate organelles from each other, that's what happens in the next step!
Step 3: Ultracentrifugigation
Now you have a solution containing lots of different organelles. Ultracentrifugigation is used to separate all these organelles:
- Cell fragments are put in a tube and spun in a centrifuge at a low speed. The heaviest organelles (e.g. nuclei) are flung to the bottom of the tube. This sediment is separated from the supernatant.
- The supernatant is spun again at a higher speed. The next heaviest organelles go to the bottom. Again, the sediment is separated from the supernatant.
- This process continues at progressively higher speeds until all organelles are separated out.
The organelles in order of mass are:
Nuclei, mitochondria, lysosomes, ER then ribosomes.