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BIOL 2321 - Cell Bio Lab > Cell fractionation > Flashcards

Cell fractionation Flashcards

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1
Q

What does it mean for a technique to be preparative?

A

it is a technique that isolates and purifies an organelle/macromolecule in sufficient quantities to be studied

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2
Q

What does it mean for a technique to be analytical?

A

the process of separating and analyzing organelles or macromolecules itself is what determines some chemical, physical, or functional property of the isolated component

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3
Q

Is cell fractionation preparative or analytical?

A

preparative

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4
Q

What is cell fractionation?

A

the isolation and purification of specific cell components

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5
Q

What are the 2 basic steps of cell fractionation?

A
  1. cellular components are released from the cell

2. the components are sorted from one another

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6
Q

What is homogenization?

A

the first step of cell fractionation:

disruption of the tissue and/or cell integrity to release the cellular contents

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7
Q

What is the result of homogenization?

A

a mixture called the homogenate which consists of organelles and macromolecules that differ from one another in their chemical and physical properties

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8
Q

What is a homogenate?

A

a mixture of organelles and macromolecules that have been released from a cell or tissue via homogenization (disruption)

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9
Q

Briefly describe the separation step of cell fractionation

A

the second step that uses the differences in physical and chemical properties of released organelles and macromolecules in the homogenate to separate them from one another

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10
Q

How is separation achieved?

A

centrifugation

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11
Q

What is a major challenge to homogenization?

A

methods that can disrupt the ECM and plasma membrane (to release cellular components) can also break up the internal membranes and destroy organelles or macromolecules

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12
Q

Why is it important to choose the right homogenization method?

A

some procedures can also destroy the internal membranes and therefore the organelles or macromolecules of interest so the right technique should be chosen to avoid this

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13
Q

What are the 3 broad categories of homogenization?

A

mechanical

sonical

chemical

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14
Q

Describe mechanical homogenization

A

disruption of cells by using physical or shearing force on cells suspended in cold buffered isotonic solution

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15
Q

What are 3 examples of mechanical homogenization devices?

A

mortar and pestle

a blender

a cell homogenizer

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16
Q

Describe a cell homogenizer

A

A device used for mechanical homogenization that creates a shearing force that breaks apart cells and tissues

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17
Q

How does a cell homogenizer work?

A

cell suspension placed in the cylindrical glass tube that is fitted with a plunger (aka pestle)

pestle is pushed to the bottom of the tube to force the cell suspension upward through a narrow space between the wall of the tube and the pestle

creates shearing force that breaks apart cells and tissues

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18
Q

What is a disadvantage of the cell homogenizer? How can this be controlled?

A

it is not selective with what it will break up

can be controlled by adjusting the distance between the side of the tube and the pestle; adjusting the speed of rotation of pestle; adjusting the roughness of the pestle or tube surfaces

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19
Q

Describe sonication

A

a method of sonical homogenization that disrupts cells or tissues using ultrasound

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20
Q

How does sonication work?

A

a sonicator probe is immersed in the cell suspension and vibrates the fluid to break up the content

ultrasonic vibrations can range from separation of cells from the tissue, breaking open the cells, to disruption of organelles

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21
Q

How can sonication be adjusted so that organelles are not destroyed?

A

adjusting the vibration frequency or exposure time to the ultrasound vibration

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22
Q

Describe chemical homogenization

A

a method of disrupting cells that involves proteolytic and/or lipolytic agents (ex. proteases or lipases or detergents) to digest the plasma membrane

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23
Q

What is a disadvantage of chemical homogenization? how can this be controlled for?

A

it is not selective because organelle membranes are also composed of proteins and lipids so proteases and lipases can digest them too

this can be controlled for by adjusting the exposure time and concentration of the chemical

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24
Q

What is centrifugation?

A

the most common method of separating and isolating cellular components after homogenization

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25
Q

Describe the centrifugation device

A

a rotor with sleeve-like holders for tubes with homogenate suspensions that is run at high speeds by an electric motor which exerts a G force on the components of the homogenate and moves them through the solution

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26
Q

How does centrifugation move cell constituents in a homogenate?

A

because organelles and other constituents vary in size and density, the centrifugal force moves them through the liquid to be suspended at different rates

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27
Q

What is sedimentation rate?

A

the rate of movement of an organelle or macromolecule through a homogenate exposed to centrifugal force which depends on their size and density and the viscosity and density of the suspending solution

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28
Q

What does rate of sedimentation depend on?

A

the organelle or macromolecule’s density and size AND the viscosity and density of the suspension solution

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29
Q

Are larger and denser components expected to move more or less quickly than smaller components? Is their expected sedimentation rate high or low?

A

they will move quicker and have a higher sedimentation rate

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30
Q

Are smaller, less dense components expected to move more or less quickly than larger/denser components? Is their expected sedimentation rate high or low?

A

slower and have lower sedimentation rate

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31
Q

What are the 2 methods of separating cell components with centrifugation?

A

differential velocity centrifugation

density gradient centrifugation

32
Q

when do you use differential velocity centrifugation for separation?

A

when the differences in sedimentation rates of the particles to be separated are large

AND

the density of the components of interest are greater than the suspension solution

33
Q

What is the result of the first centrifuge spin of the differential velocity technique?

A

the larger and denser components of interest will sediment out at the bottom of the centrifuge tube first = a pellet

the smaller, less dense components will remain in the solution = supernatant

34
Q

What happens after the first pellet and supernatant are formed in differential velocity centrifugation?

A

they are separated and the supernatant is spun again at a higher speed for longer to form a pellet and supernatant

this is repeated

35
Q

What happens to the pellet with each repetition of centrifuging the supernatant in differential velocity?

A

the resulting pellet is composed of different cellular components which become smaller and less dense each time

36
Q

Describe density gradient centrifugation

A

the sample is placed on top of a solution that increases in solute concentration as you move from the top of the tube to the bottom

as the solute concentration increases, so does the density of the solution (the gradient)

the sample and density gradient are centrifuged ONCE

37
Q

How many times are samples spun in density gradient centrifugation vs differential velocity?

A

density gradient = ONCE

differential velocity = MANY

38
Q

How are specific components isolated in the density gradient centrifugation?

A

the sample will be centrifuged in a particular density gradient sample, at a particular G value and for a particular time

39
Q

What is the result of density gradient centrifugation?

A

a series of bands (may/may not be visible) that contains components of the same size/density

40
Q

In density gradient centrifugation, will larger/denser or smaller particles travel faster?

A

larger and denser particles will travel faster

41
Q

How are the resulting bands of density gradient centrifugation arranged?

A

from smaller, less dense components forming bands at the top of the tube to larger, denser components forming bands further down the tube

42
Q

What are the 2 types of density gradient centrifugation?

A

rate sedimentation centrifugation

isopycnic centrifugation

43
Q

When is the density gradient centrifugation process stopped? What does this prevent?

A

stopped when some or all of the densities of the sample components of interest are greater than the range of the density gradient itself

prevents the components of interest from pelletizing

44
Q

What do the positions of the bands produced by density gradient centrifugation depend on?

A

the size of the structures more than the density

45
Q

Describe rate sedimentation centrifugation

A

a type of density gradient centrifugation that isolates a cell fraction according to how fast it travels through the density gradient

46
Q

How do chloroplasts move through a density gradient centrifugation vs mitochondria? What type of density centrifugation are they exposed to?

A

chloroplasts and mitochondria have the same density, but because chloroplasts are larger organelles, they will travel through the density gradient faster and appear lower than mitochondria

rate sedimentation centrifugation

47
Q

What is isopycnic centrifugation?

A

a type of density gradient centrifugation that causes components to travel to the region of the gradient with matching densities

48
Q

When is isopycnic centrifugation used?

A

used when the densities of the sample components of interest are LESS than the highest density of the gradient

49
Q

What effect will continued centrifugation have on a cell component that has reached a position in a density gradient that matches its density (isopycnic)?

A

no effect. Once a component reaches its matching density region, there will be no further movement with further centrifugation

50
Q

What is the result of isopycnic centrifugation?

A

bands in different positions from each other throughout the density gradient in the tube because the components will have different densities from each other

51
Q

How will the chloroplast band compare to a nucleus band in isopycnic centrifugation? why?

A

the chloroplast band will be in a higher band than the nucleus band because chloroplasts are less dense than nuclei, even though they are the same size

52
Q

What property of cell components does rate sediment centrifugation separate?

A

SIZE

density doesn’t matter

53
Q

What property of cell components does isopycnic centrifugation separate?

A

DENSITY

size doesn’t matter

54
Q

In an isopycnic centrifugation, where would you expect to see chloroplasts vs. ribosomes?

A

chloroplasts are less dense than ribosomes so they would be above ribosomes

55
Q

In an isopycnic centrifugation, where would you expect to see chloroplasts vs. mitochondria?

A

in the same band because they have the same densities

56
Q

In rate sedimentation centrifugation, where would you expect to see chloroplasts vs. nuclei?

A

chloroplasts will be in the same band because they are the same size

57
Q

In rate sedimentation centrifugation, where would you expect to see mitochondria vs. nuclei?

A

mitochondria will be higher than nuclei in the density gradient because mitochondria are smaller than nuclei

58
Q

What are two common density gradient solutions?

A

cesium chloride

sucrose

59
Q

Why is cesium chloride a good choice as a density gradient solution?

A

it will form a continuous density gradient when centrifuged at high speeds for long periods of time without any extra preparation

60
Q

Why is sucrose a good choice as a density gradient solution?

A

it is inexpensive and produces a stable density gradient when prepared properly

61
Q

What are the 2 types of density gradient preparations?

A

continuous density gradient

discontinuous density gradient

62
Q

What type of density gradient preparation would a rate sedimentation centrifugation use?

A

continuous density gradient

63
Q

What type of density gradient preparation would an isopycnic centrifugation use?

A

either continuous or discontinuous

64
Q

Between cesium chloride and sucrose, which would be the better option for rate sedimentation centrifugation? why?

A

cesium chloride because it creates a continuous density gradient and maintains it with multiple spins and for different time periods

65
Q

Between cesium chloride and sucrose, which would be the better option for isopycnic centrifugation? why?

A

either because isopycnic centrifugation can be done with continuous or discontinuous density gradients

66
Q

How is a discontinuous gradient made?

A

by layering solutions of different concentrations on top of one another (what we did with the sucrose gradient) to create a series of discrete bands of increasing concentration and density from top to bottom

67
Q

T or F: a continuous density gradient has discrete layers of differing concentrations and densities

A

false! that would be discontinuous

continuous gradients increase in concentration and density smoothly from top to bottom

68
Q

Normally sucrose makes a ____ density gradient, but it can be ____ and ____ to make a _____ density gradient

A

normally sucrose makes DISCONTINUOUS gradient, but it can be FROZEN and THAWED tot make a CONTINUOUS gradient

69
Q

How can sucrose be prepared to make a continuous density gradient?

A

it can be frozen and thawed

70
Q

Describe how sucrose can be used to make a continuous density gradient

A

a tube is filled with 30% sucrose and frozen = crystals of water form which exclude most of the sucrose content, leaving an unfrozen concentration of sucrose

the first crystals formed will be less dense, but as they go down the tube they will include more sucrose and become denser and denser

resulting in the upper levels of the tube having lower sucrose than the bottom

the gradient is maintained in a liquid state when it is thawed

71
Q

How would a nucleus be isolated?

A
  1. homogenization

2. isopycnic centrifugation would isolate nuclei best because they have a different density from other organelles

72
Q

How would a mitochondria be isolated?

A
  1. homogenization
  2. rate sedimentation centrifugation would isolate mitochondria from other organelles because it has a different size than the rest
73
Q

In the lab, what organism was used?

A

Tradescantia leaves

74
Q

in the lab, what technique was used to homogenate the plant tissue? Describe the method

A

mechanical homogenization

Tradescantia leaves were cut and ground in a buffer solution

75
Q

in the lab, what density gradient solution was used?

A

continuous sucrose density gradient made by freezing and thawing

76
Q

in the lab, what type of centrifugation was used to separate the cellular components of the Tradescantia leaves?

A

isopycnic centrifugation with a continuous sucrose density gradient

(separated them by density)

77
Q

In the lab, what would the position of the chloroplast band have been if the time of the centrifugation was doubled? Explain

A

in the lab we used isopycnic centrifugation so the chloroplasts will have moved to the region of the density gradient that matches their density and further centrifugation will not have had an impact

BIOL 2321 - Cell Bio Lab (10 decks)

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