Cell Fractionation

Question 1

The 3 steps:

Answer 1

Homogenisation - breaking up the cells
Filtration - getting rid of big bits
Ultracentrifugation - seperating the organelles

Question 2

Homogenisation

Answer 2

(Breaking up the cells)
- via grinding cells in a blender or vibrating cells - this breaks up plasma membrane and releases the organelles into solution
- The homogenised cell solution must be kept ice cold - to reduce enzyme activity which breaks down organelles.
+ needs to be isotonic - must have same conc of chemical as the cells broken down to prevent damage to organelles through osmosis
- a buffer solution also added to maintain the pH

Question 3

Filtration

Answer 3

(getting rid of big bits)

• via a guaze to seperate large cell debris/tissue debris from the organelles
• organelles are much smaller than debris so they pass through the gauze

Question 4

Ultracentrifugation

Answer 4

(seperating the organelles)

• cell fragments poured into tube + put into a centrifuge + spun at a low speed.
• heaviest organelle e.g. nuclei - flung to the bottom of tube + form a thick sediment (the pellet) + rest of organelles stay suspended in the fluid above (the supernatent)
• the supernatent is drained off into another tube + spun in the centrifuge at a higher speed.
• Again - heaviest goes to the bottom, form a pellet, supernatant drained off + spun at higher speed in centrifuge - its repeated until all organelle are separated.