Chop up fresh liver tissue in ice cold,
isotonic buffer solution
Put the chopped tissue in to a blender
or homogeniser to break open the cells
Filter the mixture to remove the debris
Pour the mixture in to tubes and spin
very quickly in a centrifuge
The liquid layer on top (the
supernatant) is poured in to a fresh
tube, leaving the sediment behind,
which contains nuclei.
The supernatant may then be spun
again at a faster speed to produce a
sediment containing mitochondria, and
at an even higher speed for other
organelles
Chop up fresh liver tissue in ice cold,
isotonic buffer solution
Put the chopped tissue in to a blender
or homogeniser to break open the
cells
Filter the mixture to remove the
debris
Pour the mixture in to tubes and spin
very quickly in a centrifuge
The liquid layer on top (the
supernatant) is poured in to a fresh
tube, leaving the sediment behind,
which contains nuclei.
The supernatant may then be spun
again at a faster speed to produce a
sediment containing mitochondria,
and at an even higher speed for other
organelles
Cell Fractionation
Cell fractionation, involving differential centrifugation, is a technique that allows large
quantities of an organelle to be isolated from a cell, so its function can be studied.
1. Chop up fresh liver tissue in ice cold, isotonic
buffer solution. The ice stops enzyme activity
as proteases could damage organelles. The
solution is isotonic to stop osmotic damage to
organelles and the buffer maintains a constant
pH.
2. Put the chopped tissue in to a blender or
homogeniser to break open the cells.
3. Filter the mixture to remove the debris, such
as connective tissue and plant cell walls.
4. Pour the mixture in to tubes and spin very quickly
in a centrifuge.
5. The liquid layer on top (the supernatant) is
poured in to a fresh tube, leaving the sediment,
also known as a pellet, behind. The first
sediment contains nuclei as this is the densest
organelle.
6. The supernatant may then be spun again at a
faster speed to produce a sediment containing
mitochondria and at an even higher speed for other
organelles (in order: lysosomes, rough ER, plasma
membrane, smooth ER and lastly, ribosomes).
Pour the mixture into tubes and spin
at a low speed in a centrifuge.
Put the chopped tissue in to a blender
or homogeniser to break open the cells.
Filter the mixture to remove the
debris, such as connective tissue and
plant cell walls.
The liquid layer on top (the
supernatant) is poured in to a fresh
tube, leaving the sediment, also known
as a pellet, behind. The first sediment
contains nuclei as this is the densest
organelle.
The supernatant may then be spun
again at a faster speed to produce a
sediment containing mitochondria and
at an even higher speed for other
organelles (in order: lysosomes, rough
ER, plasma membrane, smooth ER and
lastly, ribosomes).
Chop up fresh liver tissue in ice cold,
isotonic buffer solution.
The ice stops enzyme activity as
proteases could damage organelles.
The solution is isotonic to stop osmotic
damage to organelles.
The buffer maintains a constant pH.