1 Cells - Cell centrifugation/ fractionation

Question 1

What is step 1?

Answer 1

Homogenisation
- tissue sample placed in an ice cold isotonic buffer
- ice cold = stop enzyme action
- isotonic = to prevent cell from bursting due to differing water
potentials
- buffer = to prevent organelles and proteins from becoming
denatured
this breaks up the tissue to break open cells and release organelles.

Question 2

What is step 2?

Answer 2

Filtration
- removes large debris as this will be heavy and sink to the bottom of the test tube.

Question 3

What is step 3?

Answer 3

Centrifuging
- faster spinning = greater force , slower speeds mean larger fragments collect at the bottom and form a pellet. The rest of the organelles stay suspended in the supernatant. Remaining supernatant is spun again at higher speeds where another pellet containing smaller organelles is formed, the more times this process is repeated, the smaller organelles collected in pellets.

Question 4

What is the supernatant

Answer 4

The liquid in which smaller organelles are suspended after centrifuging.

Question 5

What are the purpose of the conditions during homogenisation?

Answer 5

• ice cold = stop enzyme action
• isotonic = to prevent cell from bursting due to differing water potentials
• buffer = to prevent organelles and proteins from becoming denatured