Cell Fractionation Flashcards
Why would you need to use cell fractionation ?
To view organelles under electron microscope.
Cell fractionation separates them from rest of cell sample
First step in cell fractional
Homogenisation - breaking up cells
How can homogenisation be done ?
Vibrating or grinding cells up in blender.
Why do cells need to be grinded up?
Break plasma
Cause organelles to release into solution
Name 3 things the solution must be / need ?
ICe Cold
Isotonic
Buffer added
Why ice cold ??
Reduce enzyme activity
Why isotonic ??
Same concentration of chemicals as the cells being broke down to prevent damage to organelles through osmosis.
Why buffer added ??
Maintain pH.
Second step in cell fractionation .
Filtration - getting rid of big bits
Why is homogenised solution filtered?
Through a gauze to separate large cell debris like connective tissue
Why is a gauze used ?
As organelles are much smaller than Debris so pass through gauze .
Third step in cell fractionation
Ultracentrifugation- separating organelles
Cell fragments are poured into a tube. Where does this go?
Centrifuge and spun at low speed.
What comes our first from centrifuge ?
Heaviest organelles like nuclei.
Flung to borrow of tube.
What do the organelles form at bottom of tube ?
Thick sediment at bottom called the pellet.
Rest of organelles stay suspended in fluid above - supernatant
Supernantant is drained off and poured into another tube. What happens next?
Spun in centrifuge at higher speed.
Heaviest organelles form pellet at bottom of tube .
The process repeats…
At higher and higher speeds until all organelles are separated out l.
So what happens to the pellet ?
It’s made up of lighter and lighter organelles
What is the order in mass of organelles from heaviest to lightest?
Nuclei (Chloroplast) Mitochondria Lysosomes ER Ribosomes
What is the acronym to remember the Order of organelles?
Naughty clever monkeys like eating red raspberries.
