cell fractionation Flashcards
what is cell fractionation
the separation of different parts of the cell
why do we use cell fractionation
to break down cells and their organelles to study their structure and function
what is step 1 in cell fractionation
homogenisation
what happens in homogenisation
- break cells with high frequency sounds
- use a mild detergent to make holes in the plasma membrane .
- force cells through a small hole using high pressure.
- shear cells between a close-fitting rotating plunger and the thick walls of a glass vessel
what is left behind after homogenisation
most membrane bound organelles
what is step 2 in cell fractionation
ultra centrifugation
what are the requirements for ultra centrifugation
COLD, BUFFERED and ISOTONIC
what happens in ultra centrifugation
- the tissue is grounded into smaller pieces (HOMOGENISED) by a blender
- homogenate is filtered to remove any whole cells and large debris.
- the suspension of the homogenate is then placed in a centrifuge tube and centrifuged a number of times.
- at slower speeds the higher density fragments and organelles collect at the bottom of the tube(sediment pellet) and the lower density ones remain at the top called the SUPERNATENT.
- the pellets are then removed and the supernatent is spun at higher speeds forming a new pellet at the bottom.
- continue this to get smaller and smaller fragments
what is in pellet 1
whole cells, nuclei and cytoskeletons
what is in pellet 2
mitochondria, lysosomes an peroxisomes
what is in pellet 3
microsomes and other small vesicles
what is in pellet 4
ribosomes, viruses and large macromolecules
why must it be cold
to reduce enzyme activity
why must it be isotonic
to prevent the process of osmosis
why must it be buffered
to maintain a constant PH
