cell fractionation Flashcards
(12 cards)
what is the first step of cell fractionation?
homogenisation
what happens during homogenisation?
● cold, isotonic buffer solution in test tube with tissue / test sample
● cell membranes are broke apart in a homogeniser and organelles are released
why is the buffer solution cold?
reduces enzyme activity
why is the buffer solution isotonic?
● prevents osmosis
● prevents cell bursting (lysis)
why is a buffer solution used?
● maintains pH (stops enzymes / proteins denaturing)
what is the second step of cell fractionation?
filtration
what happens during filtration?
● homogenised solution is filtered
● residue is unbroken cells (cell debris)
● filtrate is a mixture of organelles
what is the third step of cell fractionation?
ultracentrifugation
what happens during ultracentrifugation?
● organelles are separated by density in a centrifuge
● spins slow at first to separate nucleus (heaviest goes to the bottom)
● spin it faster to get the next organelle
● supernatant spun faster and longer so smaller organelles go to the bottom
what is the supernatant?
● liquid
● all organelles other than nucleus
what is the order of heaviest to smallest?
● cell wall
● nucleus
● chloroplast
● mitochondria
● ribosomes
describe and explain how cell fractionation and ultracentrifugation can be used to isolate mitochondria from a suspension of animal cells
● cell homogenisation to break open cells
● filter to remove cell debris
● use isotonic solution to prevent damage to mitochondria / organelles
● keep cold to prevent / reduce damage by enzymes / use buffer to
prevent protein / enzyme denaturation
● centrifuge to separate heavy organelles
● re-spin supernatant at higher speed to
get mitochondria in pellet / at bottom
