Cell Fractionation

Question 1

What is cell fractionation?

Answer 1

The process where cells are broken up and the different organelles they contain are separated out.

Question 2

What 3 things should the solution the tissue is placed in be? Why?

Answer 2

1. Ice cold - to reduce enzyme activity that might break down/ digest the organelles
2. Same water potential as the tissue(isotonic) - prevent organelles bursting/lysis or shrinking as a result of osmosis
3. Buffered - so the pH does not fluctuate. Any change in pH could alter the structure of the organelles or affect the functioning of enzymes by denaturing proteins

Question 3

Explain in detail the two stages of cell fractionation

Answer 3

1. Homogenisation
- cells are broken up in a homogeniser (in a cold, buffered, isotonic solution)
- this breaks the plasma membrane and releases the organelles from the cell
- homogenate (resultant fluid) is filtered to remove any complete cells and large pieces of debris

2. Ultracentrifugation
- process by which fragments in the filtered homogenate are separated in a centrifuge
1) tube of filtrate placed in centrifuge and spun at a slow speed
2) dense organelles form a pellet at the bottom of tube
3) supernatant (fluid in tube) is removed and pellet collected for study
4) supernatant transferred to another tube and spun in centrifuge at a faster speed and for a longer time
5) next dense organelle is collected in pellet 2
6) process is continued in this way so that at each increase in speed the next dense organelles is separated

Question 4

What organelles would be in each pellet?

Answer 4

Pellet 1 - nuclei
Pellet 2 - mitochondria, chloroplasts
Pellet 3 - ER/golgi apparatus
Pellet 4 - ribosomes