Lecture 11 Flashcards
What are the types of analytical systems?
Cultured cells “in vitro”
Semi permeabilized cells “in-vitro” (filter paper used to tear the membrane)
Cell-free systems “in vitro” (using organelles themselves
Reconstituted systems (from purified components of a cell)
What is the limiation of using light microscopy?
Light microscopy has limits of resolution (200nm)
What are some types of microscopy that can be used to observe cells?
Light microscopy
Video microscopy (watching live cells)
Electron microscopy
What are the steps of cell fractionation?
1) Cell can be disrupted by shear (tearing forces), sonication (sound methods of destruction), and osmotic shock (using osmotic pressure)
2) centrifugation to purify the organelles. (velocity sedimentation or equilibrium sedimentation)
3) Organelle purification by immuno isolation (
What is velocity sedimentation?
Velocity sedimentation relies on size and density. And rate of centrifugation is modified based on what organelle is being seeked.
What is equilibrium density sedimentation?
Use of density only to separate organelles (more proteins and lipid means more dense). cells are spun very fast.
What are some methods to assess purity of a centrifuged mixture?
Purity of organelles can be assessed via electron microscopy.
Another method is via a SDS gel.
What happens to more dense organelles in a centrifuge?
More dense components would go deeper down the test tube being centrifuged.
How can antibodies and protein A be used to purify organelles?
Antibodies can be used as well along with staph aureus and protein A. This mixture is centrifuged and would pallet very quickly due to the weight of the staph aureus.
How can radioactive labelling of amino acids assist with organelle purification?
Labelling of proteins with radioactive amino acids allows us to purify certain organelles from the centrifuged test tube.
How are cargo proteins detected in trafficking assays?
1) Detection of passenger proteins can be done via antibodies.
2) Fusion to reporter (GFP)
3) radiolabelling and autoradiography (following proteins via several strategies)
Why are animal cells hard to genetically modify?
Animal cells are diploid so it’s hard to modify genes in animal cells
Why are yeast the model eukaryotic organism?
Easily available
Easy to grow
Can divide in the haploid state indefinitely.
Has a small genome.
How are yeast mutants created?
1) Devise a screen to separate the mutation required from the rest.
2) Induce mutations.
3) Apply the screen to the mutants
4) Perform assays to check for the pathways step that is defective.
