Cell Fractionation

Question 1

What are the steps of cell fractionation?

Answer 1

1. Homogenisation
2. Filtration
3. Ultracentrifugation

Question 2

Describe homogenisation

Answer 2

• break down cells to release organelles by vibrating cells or grinding cells in a blender
• conditions of solution: ice cold, isotonic, buffered

Question 3

What are the 2 methods to break plasma membrane and release organelles?

Answer 3

1. Vibrating cells
2. Grinding cells in a blender

Question 4

What are the 3 conditions of the homogenised solution?

Answer 4

1. Ice cold
2. Isotonic
3. Buffered

Question 5

Why should the homogenised solution be ice cold?

Answer 5

Slow down enzyme activity to prevent denaturing

Question 6

Why should homogenised solution be isotonic?

Answer 6

Water potential of the solution should be the same as the cells that have been broken down to prevent osmosis and therefore damage

Question 7

Why should the homogenised solution be buffered?

Answer 7

Ensure pH stays constant so proteins are not denatured

Question 8

Describe the process of filtration

Answer 8

• homogenised solution filtered through a gauze to remove tissue and cell debris
• organelles are much smaller than debris so aren’t filtered out
• leaves filtrate containing mixture of organelles

Question 9

Describe the process of ultracentrifugation

Answer 9

• filtrate poured into tube and put into centrifuge and spun at low speed
• supernatant drained off and poured into another tube and spun at higher speed
• process repeated at increasing speeds until all organelles are separated

Question 10

What are pellets?

Answer 10

The heaviest organelle settled at the bottom of the tube

Question 11

What is the mass order from pellet 1-4?

Answer 11

1. Nuclei
2. Mitochondria and chloroplast
3. Lysosomes
4. Endoplasmic reticulum and ribosomes