studying cells cell fractionation

Question 1

1.What is the purpose of cell fractionation?

Answer 1

To obtain a sample of a given organelle for study in the lab.

Question 2

2. Give an overview of cell fractionation.

Answer 2

Cells are broken up (homogenisation) to release organelle, the debris is removed (filtration), and the organelles are separated using ultracentrifugation.

Question 3

3. Describe how cells are homogenised.

Answer 3

The cell membrane is broken using a blender, releasing the organelles into the extraction solution.

Question 4

4. Describe and explain the composition of
   the extraction solution.

Answer 4

ice-cold – Slows or stops enzyme activity to prevent digestion of organelles; Buffered – Maintains pH so that enzymes or proteins are not denatured; Same water potential as organelles– Prevents osmosis of water into/out of ORGANELLE so no bursting or shrinking of ORGANELLE (reject cell)

Question 5

5. Explain why the homogenised cells need to be filtered.

Answer 5

To remove any cell debris or unbroken cells.

Question 6

6. Describe and explain how organelles can be separated using ultracentrifugation.

Answer 6

The filtrate is placed in a test tube and spun at the lowest speed. The largest/heaviest/most dense organelles form a thick sediment at the bottom of the tube called a pellet. The supernatant, which is the fluid where all the other organelles are suspended, is poured off, and spun again at a higher speed. The next largest organelles form the pellet. This is repeated at progressively higher speeds until all the organelles are separated.

Question 7

7. Give the order that cell organelles will
   appear in the pellet at successively higher
   speeds.

Answer 7

Nucleus, mitochondria and chloroplasts, lysosomes, ribosomes

Question 8

8. Explain how the organelles are separated by ultracetrifugation.

Answer 8

The organelles are separated on their mass, size and density. The largest/most dense/most massive are in the first pellet when a low spin speed was used.