Separating Cell Components

Question 1

Explain the process of Cell Fractionation + Ultracentrifugation

Answer 1

1) Tissue homogenised in a homogeniser which breaks open the cells to release organelles. The solution:
- Ice cold to reduce enzyme activity
- Isotonic - Stop osmotic damage/bursting as there is no net movement in/out of organelles
- Buffer - keep pH constant

2) Homogenate is filtered to remove debris like cells not broken down
3) The filtrate is centrifuged and a pellet is formed at the bottom and the liquid above is the supernatent. First pellet is the most largest,dense organelle = nuclei
4) Supernatent poured off, respun at a faster speed for a longer time which will form 2nd most densest = chloroplast if plant, otherwise mitochondria
5) Process repeats