Cell fractionation & ultracentrifugation

Question 1

What is cell fractionation?

Answer 1

A method used to break open cells to isolate different organelles to study them.

Question 2

What are the steps for cell fractionation

Answer 2

1. Homogenisation, the cells are cut open and placed in the cold, isotonic and buffered solution. The solution is then filtered to remove large cell debris.
2. Ultracentrifugation, the filtered solution is spun at high speeds in a centrifuge. This separates the organelles according to their density.
   - The centrifugal forces cause pellets of the most dense organelle to form at the bottom of the tube.
   - The process is repeated at increasingly faster speeds, removing the supernatant each time (the liquid at the top) and leaving behind the pellet (isolated organelles)

Question 3

What is the order of organelle fractionation?

Answer 3

1. Nuclei
2. Chloroplast
3. Mitochondria
4. Lysosomes
5. Endoplasmic Reticulum
6. Ribosomes

Question 4

Why must the cells be kept in a cold, isotonic & buffered condition?

Answer 4

Cold: to reduce enzyme activity, open enzymes could damage organelles
Isotonic: to prevent osmotic damage to organelles
Buffered: to prevent enzyme denaturation

Exam tip - always refer to ‘reducing damage to organelles’ if these conditions are kept.