Cell Fractionation

Question 1

What is cell fractionation

Answer 1

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

Question 2

What are the two stages

Answer 2

• homogenisation- breaking up cells
• ultracentrifugation- separating separate components

Question 3

Why is the tissue placed in a cold isotonic buffer

Answer 3

Cold- reduce enzyme activity which may break down organelles
Isotonic- (water potential same in and out of cell) prevents organelles bursting or shrinking due to osmotic gain or loss of water
Buffer- to maintain a constant pH to prevent damage to organelles

Question 4

Stages of cell fractionation

Answer 4

1) chop up fresh tissue in cold isotonic buffer solution
2) put tissue in a blender or homogeniser to breakdown cells
3) filter the mixture to remove debris such as connective tissues and cell walls
4) pour mixture into tubes and spin quickly in a centrifuge (densest at the bottom)
5) the supernatant (liquid layer) is poured into a fresh tube leaving the pellet (nucleus) behind
6) supernatant spun at a quicker speed to produce a sediment containing mitochondria. It is then spun even quicker for other organelles

Question 5

What is the order of the organelles in stage 6 of cell fractionation

Answer 5

Lysosomes, rough ER, plasma membrane, smooth ER and ribosomes

Question 6

What is the process of centrifuging at different speeds called

Answer 6

Differential centrifugation