Cell fractionation and ultracentrifugation

Question 1

What is the purpose of cell fractionation?

Answer 1

To separate cell components so that they can be studied in detail

Question 2

What are the 4 steps of cell fractionation?

Answer 2

• Sample preparation
• Homogenisation
• Filtration
• Ultracentrifugation

Question 3

What happens in step 1 ‘sample preparation’?

Answer 3

The sample in placed in a cold, isotonic, buffered solution

Question 4

What is the purpose of the solution being ‘cold’?

Answer 4

Prevents enzyme activity so that organelles do not get broken down

Question 5

What is the purpose of the solution being ‘isotonic’?

Answer 5

Ensures that the water potential inside and outside the cells are the same which prevents osmotic lysis occurring in organelles

Question 6

What is the purpose of the solution being ‘buffered’?

Answer 6

Maintains constant pH to ensure that the tertiary structures of the organelles remain the same

Question 7

What happens in step 2 ‘homogenisation’?

Answer 7

Cells are broken open as the plasma membrane is disrupted and organelles are released

- Cells are blended in the cold, isotonic, and buffered solution

Question 8

What happens in step 3 ‘filtration’?

Answer 8

The mixture is filtered to remove larger pieces of debris and any remaining tissue fragments

Question 9

What happens in step 4 ‘ultracentrifugation’?

Answer 9

1) The cell fragments are placed in a centrifuge tube and spun at a low speed
2) This results in the heaviest organelles, such as nuclei, forming a pellet at the bottom of the tube
3) The lighter organelles remain suspended in the supernatant
4) The supernatant is then transferred to a new tube and centrifuged at a higher speed
5) This leads to the next heaviest set of organelles, typically mitochondria, settling into a pellet
6) Steps 4 and 5 are repeated, increasing the speed each time to separate remaining organelles

Question 10

What is meant by ‘pellet’?

Answer 10

The sediment at the bottom of the tube, containing the heavier organelles

Question 11

What is meant by ‘supernatant’?

Answer 11

The liquid remaining above the pellet, containing the lighter organelles

Question 12

State the order of organelles from heaviest to lightest

Answer 12

Nuclei (heaviest)
Chloroplasts
Mitochondria
Lysosomes
Endoplasmic reticulum
Ribosomes (lightest)

Question 13

What is ultracentrifugation?

Answer 13

The process used to separate organelles based on their densities
- By being spun at different speeds, organelles separate into layers according to their mass