Cell Fractionation

Question 1

What is cell fractionation?

Answer 1

Process where cells are broken up and the different organelles are separated out.

Question 2

What must you do before cell fractionation?

Answer 2

• Place the tissue in a cold, buffered solution with the same water potential
• Coldness, reduces enzyme activity
• Buffered, pH doesn’t fluctuate
• Same water potential, prevents organelles or shrink as a result of osmosis

Question 3

Steps of cell fractionation

Answer 3

Step 1 - Homogenisation (Breaking up cells):
- Use a blender (homogeniser)
- Plasma membrane broken, so organelle go into solution
This is called homogenate
Step 2 - Filtration ( Remove complete cells or debris)
- Filter thorugh a gauze
- Filter doesn’t separate organelle
Step 3 - Ultracentrifugation (Separating the organelle)
- Mixture of organelle placed in a centrifuge
- First spin homogenate at a low speed, this results in the heaviest organelle sinking( nuclei) leaving a supernatant.
- Second spin of supernatant at medium speed (next heaviest organelle creates sediment at bottom)
- Third spin is spun at a high speed so the lightest-heaviest organelle sinks

Question 4

Order that organelle come out

Answer 4

Nucleus
Chromosomes
Mitochondria
Lysosomes
Endoplasmic Reticulum
Ribosomes