Homogenisation is the breaking up of cells. This can be done in several ways either by vibrating the cells of by grinding up the cells. Homogenisation breaks up the plasma membranes and releases the organelles into the solution.
Flitration is getting rid of larger parts of the cell, this is done by passing the homeogenised cells through a gauze. Flitering the solution gets rid of any cell debris or tissue debris, this is because organelles are much smaller than the debris so the organelles pass through the gauze.
Ultracentrifugation is the seperating of organelles. After filtration you are left with a solution of different organelles. In order to seperate any particular organelles you have to carry out ultracentrifugation. This is done by-
- Cell fragments are poured into a test tube.
- Test tube put in a centrifuge (a machiene that seperates organelles) it is spun on a low with a force of around 10000g (g, force that is so many times gravity) for 15-20 mins.
- The spinning motion causes the heaviest organelles to sink to the bottom to form a thick sediment and then pellets.
- The rest of the soloution is liquid this is called supernatant.
- Supernatent is drained off and poured into another test tube and put in the centrifuge again at a higher speed and the heaviest organelles form pellet in the bottom of the test tube
- This processes is then repeated until all the organelles are seperated.
Requirements of solution for Ultracentrifugation
The organelle solution's salt concentration is made isotonic this means that osmosis cant take place and the organelles vary in mass. The soloution is also buffered to minimise change in pH so that the enzymes in the organelles dont start to denature. The temperature of the soloution is kept low to slow down metabolism and to minimise self digestion of the organelles.