Cell Fractionation Flashcards
How do we study cells
Using light or electron microscopes
Characteristics of a light microscope
Lower magnification and resolution
Show images in colour
Characteristics of electron microscope
High magnification and resolution
Show images in black and white
What do we have to do to cells to see their cell components
Separate them
What are the stages to separating cell components
Homogenation
Filtration
Ultracentrifugation
What conditions should homegenates be placed in
Cold, buffered, isotonic solution
Why should homogenates be placed in a cold solution
Because it reduces enzyme activity (which could break down the organelles which is bad)
Why should homogenates be placed in an isotonic solution
Prevents organelle suffering from plasmolysis or lysis as a result of osmotic gain or water loss, keeping the organelle from damage
Define isotonic solution
Solution has same H2O potential as the original tissue
Why should homogenates be placed in a buffered solution
Maintains a constant pH so enzymes don’t denature
Why is enzyme activity bad for looking at cell components
Enzyme activity could break down organelles
What happens during homogenation
Disrupts the cell membrane, breaking open cells to release the organelles
Why do we filter the homegenate
To remove large unwanted debris
Eg whole cells
What happens during ultracentrifugation
The homogenate is centrifuged at a low speed
Remove the pellet of the heaviest organelle and respin the supernatent at a higher speed
Repeat at increasing speeds until til everything is separated out
Order of organelles heaviest to lightest
Heaviest - nuclei
Chloroplasts/mitochondria
Lysosomes
Endoplasmic reticulum
Lightest - ribosomes
