Methods of studying cells Flashcards
Describe what is meant by cell fractionation
The process in which organelles are separated
What are the 2 parts of cell fractionation called
- Homogenisation: cells are broken apart
- Centrifugation: the organelles are separated
What 3 conditions must our tissue samples be kept under, when carrying out cell fractionation
- Buffered
- Isotonic
- Cold
Why are tissue samples stored in a buffered solution
- Prevents PH changes, so that hydrogen and ionic bonds in the tertiary structure of proteins within organelles, remain unaffected
Why are the tissue samples stored in an isotonic solution
- Equal water potential, therefore water doesn’t move into organelles via osmosis, preventing the lysis of organelles
Why are tissue samples stored in a cold solution
- Reduces enzyme activity, which prevents the hydrolysis of proteins within organelles
Describe how cells can be homogenised
- Physically using a mortar and pestle
- Chemically using a weak detergent
- The cell membranes are disrupted, to release the organelles within
Describe what happens after homogenisation
The solution is filtered to remove unbroken cells and large pieces of debris, forming a mixture called a homogenate
Describe what happens in centrifugation
- A centrifuge separates things according to their different densities
- The homogenate is placed in a centrifuge and spun
- The most dense organelle falls to the bottom (pellet) and is removed, and the liquid on top (supernatant) will be spun at a faster speed to cause the next most dense organelle to fall to the bottom
Starting with some lettuce leaves, describe
how you would obtain a sample of undamaged
chloroplasts. Use your knowledge of cell
fractionation and ultracentrifugation to
answer this question.
- Chop up (accept any reference to crude breaking up);
- Cold;
- Buffer solution;
- Isotonic / same water potential;
- Filter and centrifuge filtrate;
- Centrifuge supernatant at higher speed;
- Chloroplasts in (second) pellet;