cell fractionation

Question 1

what is cell fractionation

Answer 1

the process where cells are broken up and the different organelles they contain are seperated out

Question 2

before c.f can begin what happens

Answer 2

the tissue is placed in a cold, isotonic, buffered solution

Question 3

why would the solution (c.f) be cold

Answer 3

to reduce enzyme activity that might break down the organelles

Question 4

why would the solution be isotonic (cf)

Answer 4

same water potential as tissue - prevents organelles bursting or shrinking as a result of osmotic gain or loss of water

Question 5

why would the solution be buffered (cf)

Answer 5

so the ph doesnt fluctuate - any change could alter the structure of the organelles or affect the functioning of the enzymes

Question 6

what is homogenation

Answer 6

cells are broken up by a homogeniser. this releases the organelles from the cell the resultant fluid, the homogenate, is then filtered to remove any whole cells and large pieces of debris.

Question 7

ultracentrifugation def

Answer 7

process where the fragments in the filtered homogenate are seperated in a machine called a centrifuge. this spins tubes of homogenate at a very high speed in order to create a centrifugal force.

Question 8

step by step ultracentrifugation

Answer 8

• the tube is placed in centrifuge and spun at a slow speed
• the heaviest organelles, the nuclei, are forced to the bottom of the tube where they form a thin sediment or pellet
• the fluid at the top of the tube is removed leaving just the sediment
• the supernatant is transferred to another tube and spun at a faster speed
• the next heaviest organelles, the mitochondria, are forced to the bottom of the tube
• this is continued and speed increases