Test 1: Analysis after Purified Culture Grown Flashcards
Cell Fractionation is used to…
purify organelles of interest.
The three steps of Fractionation is…
- Lyse
- Centrifuge purification
- Analysis
Lysing must be gentle to…
preserve organelles
Lysing can be done by… (5)
- Sonification
- Grinding
- Filtration (thru extra small holes)
- Osmotic stress (to cause lysing)
- Detergent (disrupt PM but not organelle membrane)
While lysing, ___ must be used to cushion organelles. This is to prevent ___
Buffer (0.2 M sucrose), osmotic stress
Centrifuge Purification has 3 different methods.Which is rough and which is finer?
- Differential - rough
- Equilibrium - fine
- Velocity - fine
Differential Centrifugation is separation by…
size
Differential Centrifugation is done to the homogenate. Pellet has…
whole cells, nuclei, cytoskeleton
After pellet is medium speed centrifugation. Pellet contains…
mitochondria, lysosome, peroxisome.
After medium speed centrifugation is high speed centrifugation. Pellet contains…
Microsome (from ER), small vesicles
After high speed centrifugation is very high speed centrifugation. Pellet contains…
Ribosome, virus, and large macromolecules.
Velocity Centrifugation has a (shallow/deep) gradient and sorts by…
shallow; shape and size
s-value is also called… This is for _ Centrifugation. What organelle is labeled with this?
sedimentation coefficient. Velocity. 40s v 80s ribosome.
Equilibrium Centrifugation has a (shallow/deep) gradient and sorts by…
Deep; density
In (velocity/equilibrium) centrifugation, organelle is stopped before it can match zone of density/hit bottom
velocity
Velocity of particle = …
([centrifuge force]/[frictional force]) - (1 - [density of medium]/[density of particle])
Velocity of particle = …: In Velocity Centri, what term approaches zero? Why? So what determines velocity?
Second term, because density of medium (shallow gradient) «_space;density of particle. Frictional Force (or size and shape) det velocity
Velocity of particle = …: In equi centri, density of particle and density of medium differs. If velocity is negative, density of particle is (greater than/lesser than) that of medium, the second term is (-/+) and it will move (downward/upward).
less than; -; downward
After organelle purification comes…
analysis
Ensure purity of organelles by…
- Microscopy (look as in cell, and only 1 kind)
2. Enzymatic activity (must not have other organelle specific activity AND express target organelle specific activity)
Determining which macromolecule is required for organelle function by… This is done (in vivo/in utero)
Cell Free System; in vitro
Cell Free System requires 2 steps:
- Fractionalize organelle to separate macromol
2. Add back in different combo to test for activity of interest
Complicating factors for Cell Free System are…
- Inhibitors
2. Codependent proteins
