ULTRACENTRIFUGATION Flashcards
what is cell fractionation
process where cells are broken up and the different organelles they contain are separated
helps with the study of cell structure and function
what are the 2 stages of cell fractionation
homogenation
ultracentrifugation
why a cold buffered isotonic solution
cold - reduce enzyme activity which may break down organelles
isotonic - prevents organelles bursting / shrinking due to gain / loss of water
buffered - maintain a constant pH to prevent damage to organelles
what is an ultracentrifuge
refrigerated low pressure chamber with a rotor
rotor speed can be changed slowly
steps of cell fractionation
- tissue minced and placed in a cold, isotonic, buffered solution
- a homogeniser grounds it into smaller pieces releasing the organelles from the cell membrane
- the homogenate is filtered removing complete cells and large debris
stages of ultracentrifugation
- a suspension of homogenate is placed in a test tube and centrifuged
- slower speeds - large fragments (sediment pellets0 collect a the bottom and small fragments remained at the top suspended in the supernatant liquid
- sediment pellets are removed and the remaining supernatant is respun at a faster speed at greater force. some of the smaller fragments collect at the bottom forming a new pellet
- smaller and smaller fragments can be recovered
what is differential centrifugation
process involving centrifuging at different speeds
organelle sizes are relatively constant and seem to seperate at specific speeds
process of differential centrifugation
when the centrifuge spins, the centrifugal force causes pellets of the densest organelles to form at the bottom
the centrifuge is first spun at a low speed and then the process is repeated with increasing speeds
each time, the supernatant is moved, leaving behind a pellet of organelles
