Test 1: Analysis after Purified Culture Grown

Question 1

Cell Fractionation is used to…

Answer 1

purify organelles of interest.

Question 2

The three steps of Fractionation is…

Answer 2

1. Lyse
2. Centrifuge purification
3. Analysis

Question 3

Lysing must be gentle to…

Answer 3

preserve organelles

Question 4

Lysing can be done by… (5)

Answer 4

1. Sonification
2. Grinding
3. Filtration (thru extra small holes)
4. Osmotic stress (to cause lysing)
5. Detergent (disrupt PM but not organelle membrane)

Question 5

While lysing, ___ must be used to cushion organelles. This is to prevent ___

Answer 5

Buffer (0.2 M sucrose), osmotic stress

Question 6

Centrifuge Purification has 3 different methods.Which is rough and which is finer?

Answer 6

1. Differential - rough
2. Equilibrium - fine
3. Velocity - fine

Question 7

Differential Centrifugation is separation by…

Answer 7

size

Question 8

Differential Centrifugation is done to the homogenate. Pellet has…

Answer 8

whole cells, nuclei, cytoskeleton

Question 9

After pellet is medium speed centrifugation. Pellet contains…

Answer 9

mitochondria, lysosome, peroxisome.

Question 10

After medium speed centrifugation is high speed centrifugation. Pellet contains…

Answer 10

Microsome (from ER), small vesicles

Question 11

After high speed centrifugation is very high speed centrifugation. Pellet contains…

Answer 11

Ribosome, virus, and large macromolecules.

Question 12

Velocity Centrifugation has a (shallow/deep) gradient and sorts by…

Answer 12

shallow; shape and size

Question 13

s-value is also called… This is for _ Centrifugation. What organelle is labeled with this?

Answer 13

sedimentation coefficient. Velocity. 40s v 80s ribosome.

Question 14

Equilibrium Centrifugation has a (shallow/deep) gradient and sorts by…

Answer 14

Deep; density

Question 15

In (velocity/equilibrium) centrifugation, organelle is stopped before it can match zone of density/hit bottom

Answer 15

velocity

Question 16

Velocity of particle = …

Answer 16

([centrifuge force]/[frictional force]) - (1 - [density of medium]/[density of particle])

Question 17

Velocity of particle = …: In Velocity Centri, what term approaches zero? Why? So what determines velocity?

Answer 17

Second term, because density of medium (shallow gradient) &laquo_space;density of particle. Frictional Force (or size and shape) det velocity

Question 18

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).

Answer 18

less than; -; downward

Question 19

After organelle purification comes…

Answer 19

analysis

Question 20

Ensure purity of organelles by…

Answer 20

1. 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)

Question 21

Determining which macromolecule is required for organelle function by… This is done (in vivo/in utero)

Answer 21

Cell Free System; in vitro

Question 22

Cell Free System requires 2 steps:

Answer 22

1. Fractionalize organelle to separate macromol

2. Add back in different combo to test for activity of interest

Question 23

Complicating factors for Cell Free System are…

Answer 23

1. Inhibitors

2. Codependent proteins