3.9 - Cell Fractionation

Question 1

What is cell fractionation?

Answer 1

Laboratory method isolating cell components

Question 2

What is the purpose of cell fractionation?

Answer 2

To study organelles in detail

Question 3

What are the four main steps of cell fractionation?

Answer 3

1. Sample preparation
2. Homogenisation
3. Filtration
4. Ultracentrifugation

Question 4

What type of solution is used in sample preparation?

3 Key points

Answer 4

• Ice-cold - This slows down enzyme activity that might otherwise break down organelles.
• Isotonic - It ensures that the water potential inside and outside the organelles is the same, preventing damage through water movement.
• Buffered - Keeping the pH constant is crucial to prevent denaturation of proteins and enzymes.

Question 5

Why is the solution ice-cold?

Answer 5

To slow down enzyme activity

Question 6

Why is the solution isotonic?

Answer 6

To prevent damage through water movement

Question 7

Why is the solution buffered?

Answer 7

To prevent denaturation of proteins and enzymes

Question 8

What is homogenisation?

Answer 8

Physically breaking open cells

Question 9

How is homogenisation achieved?

Answer 9

Using a blender to grind cells

Question 10

What is filtration?

Answer 10

Removing larger pieces of cell debris

Question 11

What is used for filtration?

Answer 11

The filtration is typically done through a gauze, which allows smaller organelles to pass through while retaining larger debris.

Question 12

What is ultracentrifugation?

Answer 12

Separating organelles based on density

Question 13

What is a pellet?

Answer 13

Sediment at the bottom of the tube

Question 14

What is a supernatant?

Answer 14

Liquid remaining above the pellet

Question 15

What are the heaviest organelles?

Answer 15

Nuclei

Question 16

What are the lightest organelles?

Answer 16

Ribosomes

Question 17

What is the order of organelles from heaviest to lightest?

Answer 17

1. Nuclei (heaviest)
2. Chloroplasts
3. Mitochondria
4. Lysosomes
5. Endoplasmic reticulum
6. Ribosomes (lightest)

Question 18

How are organelles separated in ultracentrifugation?

Answer 18

By spinning at various speeds

Question 19

What happens to the supernatant after each spin?

Answer 19

It is transferred to a new tube and centrifuged at a higher speed