Transcript 6 Cell Fractionation

The Ultrastructure of the Cell
Task 1 – Labelling cell diagrams
Let’s check the homework.
Task 2 – Exam questions
Cell Fractionation and
Ultracentrifugation
Cells consist of a number of organelles.
We can examine their structure and function if we
can isolate them.
Cell fractionation = breaking up a cell to release its
organelles.
Ultracentrifugation = a method for the separation
of the organelles by density.
Extracting organelles from cells
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Steps of Cell Fractionation & Ultra Centrifugation
Cell Fractionation
1. Tissue to be studied is cut into small pieces and
placed into an ICE COLD, ISOTONIC BUFFER
solution.
Why?
ICE COLD to stop enzyme activity.
ISOTONIC (same concentration/water potential
as cytoplasm) to prevent osmosis which would
damage organelles).
BUFFER to keep pH constant so proteins are not
denatured.
Steps of Cell Fractionation & Ultra Centrifugation
Cell Fractionation
2. Ground into smaller pieces using a
HOMOGENISER to release the organelles
from the cell.
3. The HOMOGENATE is filtered to remove
any complete cells and large debris i.e. cell
walls/membranes.
Steps of Cell Fractionation & Ultra Centrifugation
Ultracentrifugation
1. Homogenate is placed in a test tube and
centrifuged.
The faster the speed at which the tube is
spun, the greater the force generated.
2. Larger, more dense fragments collect at the
bottom of the tube to form a PELLET.
Smaller ones remain near the top suspended
in a liquid called the SUPERNATENT.
Steps of Cell Fractionation & Ultra Centrifugation
Ultracentrifugation
3. Pellet is removed and the supernatant
remaining is re-spun at a faster speed (more
force).
4. Process is repeated at higher and higher
speeds.
Important info!
Organelle sizes are
similar in all cells so
we can predict when
they will form a
pellet.
Since the whole
process involves
centrifuging at
different speeds it
is called
DIFFERENTIAL
CENTRIFUGATION.