2.1 Cell Fractionation and Ultracentrifugation

Question 1

What is cell fractionation?

Answer 1

The process where cells are broken up and the organelles are separated out

Question 2

What happens before cell fractionation?

Answer 2

The tissue is placed in a cold, buffered solution of the same water potential as the tissue

Question 3

Why is the tissue place in a COLD, buffered solution of the same water potential as the tissue before cell fractionation?

Answer 3

To reduce enzyme activity that might break down organelles

Question 4

Why is the tissue place in a cold, buffered solution of the SAME WATER POTENTIAL as the tissue before cell fractionation?

Answer 4

To prevent osmotic lysis (splitting)

Question 5

Why is the tissue place in a cold, BUFFERED solution of the same water potential as the tissue before cell fractionation?

Answer 5

Changes in pH could damage the organelles

Question 6

What is water potential?

Answer 6

The measure of the ability of water molecules to move freely in solution

Question 7

What happens in homogenation?

Answer 7

• cells are broken up by a homogeniser (blender)
• resultant fluid is know as the homogenate
• it is then filtered to remove any complete cells and large pieces of debris

Question 8

What is ultracentrifugation?

Answer 8

• process that separates the fragments in the homogenate
• using a machine called a centrifuge which spins at very high speeds

Question 9

What are the 2 stages to cell fractionation?

Answer 9

• homogenation
• ultracentrifugation

Question 10

How does cell fractionation work?

Answer 10

1. Tube of filtrate is placed in the centrifuge and spun at low speeds
2. Heaviest organelles (the nuclei) are forced to the bottom of the tube forming a sediment or pellet
3. The fluid at the top (supernatant) is removed
4. The supernatant is transferred to another tube and spun at a higher speed
5. The next heaviest organelles (mitochondria) are forced to the bottom of the tube
6. Each spin increases in speed and separates out the next heaviest organelle

Question 11

What are the 3 organelles that need to be separated out in cell fractionation?

Answer 11

• nuclei
• mitochondria
• lysosomes

Question 12

What is the speed of centrifugation (revolutions per minute) needed for nuclei?

Answer 12

1,000

Question 13

What is the speed of centrifugation (revolutions per minute) needed for mitochondria?

Answer 13

3,500

Question 14

What is the speed of centrifugation (revolutions per minute) needed for lysosomes?

Answer 14

16,500

Question 15

What is an isotonic solution?

Answer 15

Same water on both sides so water goes in and out at the same rate and cell will stay the same size

Question 16

What is a hypotonic solution?

Answer 16

More water on outside of cell so the cell swells and bursts - lysis

Question 17

State the order of sedimentation of organelles during differential centrifugation.

Answer 17

most dense -> least dense

nucleus, chloroplast, mitochondria, lysosomes, RER, plasma membrane, SER, ribosomes