T2: Cell Fractionation + Ulttracentrifugation

Question 1

what is cell fractionation ad what are the stages?

Answer 1

separating cell organelles from each other
breaking up tissue and then centrifuging the mixture at different speeds

Homogenisation
Filtration
Ultracentrifugation

Question 2

Describe homogenisation

Answer 2

place sample in a cold isotonic buffer solution
grind the cells up in homogeniser

this breaks the plasma membrane of the cells and releases the organelles into homogenate

Question 3

why must the tissue be put into a ice cold isotonic buffer solution before homogenising?

Answer 3

ice cold: reduce the activity of enzymes that break down the organelles

isotonic: must have the same water potential to prevent cells from bursting under osmotic pressure

buffered: to prevent organelle proteins from becoming denatured, stops pH fluctuations

Question 4

describe filtration

Answer 4

homogenate is filtered through a gauze (leaving filtrate)
to separate out any large cell debris or tissue debris that were not broken up

the organelles are much smaller than the debris so are not filtered out

Question 5

describe ultracentrifugation

Answer 5

filtrate spun in a centrifuge tube at a low speed
so heaviest organelles settle at the bottom forming a pellet

supernatant remains above
the supernatant is drained off and placed into another tube, which is spun at a higher speed

process is repeated at increasing speeds until desired organelle is separated out

each new pellet formed contains a lighter organelle than the previous pellet

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