Cell Fractionation

Question 1

What is cell fractionation

Answer 1

Separating organelles from the rest of the cell

Question 2

What are the 3 steps

Answer 2

Homogenisation-Breaking up the cell
Filtration -Getting Rid of the Big Bits
Ultracentrifugation-Separating the Organelles

Question 3

What is homogenisation

Answer 3

Vibrating cells or grinding the cells up in a blender which breaks up the plasma membrane and releases the organelles into solution

Question 4

What are the conditions for homogenisation

Answer 4

-Ice-cold-to reduce the activity of enzymes that break down organelles
-Isotonic — this means it should have the same concentration of chemicals as the cells being broken down which prevents damage to organelles through osmosis
-A buffer solution should be added to maintain the pH.

Question 5

What are the steps of filtration

Answer 5

-homogenised solution is filtered through a guaze to separate any large cell debris or tissue debris
Organelles are much smaller than the debris so they pass through the gauze

Question 6

What are the steps of ultracentrifugation

Answer 6

1) The cell fragments are poured into a tube. The tube is
put into a centrifuge and is spun at a low speed. The heaviest organelles, like nuclei, get flung to the bottom of the tube by the centrifuge. They form a thick sediment at the bottom — the pellet. The rest of the organelles stay suspended in the fluid above the sediment — the supernatant.
2) The supernatant is drained off, poured into another tube, and spun in the centrifuge at a higher speed.
Again, the heaviest organelles, this time the mitochondria, form a pellet at the bottom of the tube. The supernatant containing the rest of the organelles is drained off and spun in the centrifuge at an even higher speed.
3) This process is repeated at higher and higher speeds, until all the organelles are separated out. Each time, the pellet at the bottom of the tube is made up of lighter and lighter organelles.