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Molecular 2018 > Lecture 21 > Flashcards

Lecture 21 Flashcards

1
Q

Define the characteristics of a primary cell culture. give an example.

A

derived directly from the animal

ensymatic/mechanical treatment is used to isolate the cell of interest from a heterogenous sample

usuallys “survive for a finite period of time”

ex. primary neurons or cardiomyocytes

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2
Q

Define the characteristics of an established/continuous cell line culture. give an example

A

a primary culture that has been made immortal via transformation

Usually tumor derived or transformed with a virus

ex. SH-SY-5Y (human neuroblastoma derived), and many other cancers

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3
Q

What are some advantages of culturing cells?

A

study of cell behavior without complexity/variation that occurs in nature

Good reproducibility between experiments

Uniformity of sample

researchers have total control over exposures (to simulate diseases/study pharmacological effects)

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4
Q

what are some disadvantages of culturing cells?

A

techniques must be developed/standardized to maintain healthy cells

Quantity of material is limited

Dedifferentiation and selection may occur and impact the original cellular mechanisms/pathways

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5
Q

To create a cell model of Parkinson’s disease, you would expose _____ cells to 6-OHDA in order to create reactive oxygen species that trigger apoptosis/decrease PMCA

A

SH-SY5Y

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6
Q

what does PMCA stand for?

A

plasma membrane Ca2+ ATPase (calcium pump that uses ATP to keep the Ca2+ gradient in neurons) that pumps Ca2+ out of the neuron

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7
Q

How is 6-OHDA (6-hydroxydopamine) created?

A

dopamine is converted to 6-OHDA

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8
Q

_____ is crucial to be able to study the unique structure/function of INDIVIDUAL proteins, and this process may also use _____ DNA technology to over express a protein

A

Purification

Recombinant

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9
Q

What is commonly done before purification of a protein, in order to reduce the complexity of the material in a sample?

A

sub-cellular fractionation

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10
Q

When it comes to sub-cellular fractionation, what is homogenate?

A

suspension of different cell types (usually achieved via mechanical blending of a sample

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11
Q

When it comes to sub-cellular fractionation, give some examples of processes used to achieve lysis of cells

A

osmotic shock, ultrasonic vibration, mechanical blending, and forcing the sample through a small orifice (not important)

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12
Q

When it comes to sub-cellular fractionation, what is ultracentrifugation used to achieve?

A

the separation of organelles

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13
Q

describe the sequential process of sub-cellular fractionation

A

mechanical blending

centrifugation of the homogenate to separate based on size/density

lysis of cells

ultracentrifugation

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14
Q

what is the difference between a fixed angle rotor and a swinging bucket rotor centrifuge.

A

fixed angle: test tubes stay in the same position

Swinging bucket rotor: test tubes have a hinge at the top and swing parallel to the floor (like a helicopter)

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15
Q

describe what occurs, in terms of what collects at the bottom of the test tube, at low-speed, medium-speed, high-speed, and very-high-speed centrifugation.

A

LS: whole cells, nuclei, and cytoskeletons

MS: mito, lysosomes, and peroxisomes

HS: microsomes, and small vesicles

VHS: ribosomes, viruses, and large macromolecules

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16
Q

describe density gradient centrifugation and how low-buoyant density and high-buoyant density substances are collected in high concentrations for testing

A

low-buoyant density components collect in a line towards the top of the test tube, after centrifugation

high-buoyant density components collect in a line towards the bottom of the test tube, after centrifugation

several test tubes worth of the different buoyancy components are collected and concentrated into separate test tubes

17
Q

what happens to PMCA activity with age? how is PMCA activity inhibited?

A

it declines

reactive oxygen species (ROS)

18
Q

give a brief, overall description of column chromatography

A

a solid matrix is loaded into a tube with a porous plug at the bottom

a sample is applied to the top of the matrix from a large reservoir

the different molecules in the sample are separated by various properties by the matrix

19
Q

In terms of column chromatography, compare Ion-exchange, gel-filtration, and affinity matrices.

A

Ion-exchange: depending on the charge of the matrix beads, oppositely charged particles will stick to the bead and the rest of the sample passes through

Gel-filtration: mechanical separation where small-enough molecules become “retarded” in a porous bead

Affinity: Enzyme affinity covalently attaches enzymes to substrates, that are moving through the column, to the bead

20
Q

what type of beads would be used in affinity chromatography to separate PMCA from a sample?

A

calmodulin sepharose beads

21
Q

List the 4 techniques used to analyze proteins

A

SDS-PAGE

Western Blotting

ELISA

Mass Spectometry

22
Q

describe the purpose of SDS PAGE

A

It’s purpose is to separate proteins based on their size (smaller (lighter) moves faster and larger(heavier) moves slower)

23
Q

describe the mechanism of SDS PAGE

A

SDS is a hydrophobic and negatively charged treatment that unfolds proteins and gives all proteins in a sample a uniform charge

Beta-mercaptoethanol: reduces the disulfide bonds between the subunits in some proteins (separates subunits)

Polyacrylamide-gel electrophoresis: sample loaded at the cathode (-) end of gel and is electrophoresed towards the Anode (+) end to separate proteins

24
Q

Why is SDS, when used for SDS-PAGE, used to give all proteins present in a sample a uniform charge?

A

if this did not occur, the electrophoresis part of SDS-PAGE analyzation would separate proteins based on their size AND their various charge types and the amount of charge

it would be much more confusing than just separating based on size, so SDS makes the electrophoresis more easily interpreted

25
Q

How are the results of SDS-PAGE made to be more easily visualized?

A

staining techniques/dyes, such as coomassie blue, are applied to more easily visualize the separated bands of proteins

26
Q

What type of changes can Western Blotting identify?what type of proteins is western blotting used for?

A

Changes in the antigen and changes in the protein

known/specific proteins

27
Q

Describe the process that western blotting utilizes (6 steps)

A

the protein sample is electrophoresed onto a gel

it is “blotted” onto a membrane that immobilizes the protein sample

the membrane w/ the sample is “blocked” with a neutral protein

the primary antibody is applied and the membrane is incubated

The secondary antibody (the marking one) is applied and the membrane is incubated

Results are visualized

28
Q

Compare the function of primary and secondary antibodies in western blotting technique

A

The primary antibody binds to the immobilized antigen of interest

The secondary antibody binds to the complex of the immobilized antigen and the primary antibody to “mark” it (usually this is a flourescent substance)

29
Q

What does ELISA stand for? What does this test for?

A

Enzyme-linked Immunosorbent Assay

tests for levels (or changes in levels) of specific antigen OR antibody concentrations

30
Q

Describe what Indirect ELISA tests for. describe the order of the complex formed, starting with the immobilized portion.

A

the amount of an antibody in a sample (using conjugating antigens)

immobilized antigen; specific antibody; enzyme-linked antibody

31
Q

Describe what Sandwich ELISA tests for. describe the order of the complex formed, starting with the immobilized portion.

A

the amount of antigen in a sample (using conjugating antibodies)

immobilized monoclonal antibody; antigen ; second monoclonal enzyme-linked antibody

32
Q

Describe the process of ELISA and how it is read

A

either the antibody or the antigen of interest in the sample is quantified based on the color change observed in the well

various samples are put into wells and a known, color-changing antibody/antigen is added

(darker color change = higher concentration of substrate in the sample)

33
Q

what type of test is a pregnancy test and example of? what exactly are pregnancy tests looking for in the urine sample?

A

Sandwich ELISA test

testing for hCG, which is a hormone that is present in the urine during pregnancy

34
Q

on a pregnancy test, explain the differences between the “reaction site”, the “control site” and the “test site”

A

Control site: has an immobilized non-specific antibody that changes color when it contacts any antibody

reaction site: has free hCG antibodies that bind to any hCG present in the urine sample

Test Site: has an immobilized hCG antibody that binds to the free hCG antibody-hCG complex from the reaction site

35
Q

What is mass spectrometry used for? what are the 3 things it requires?

A

it is used to identify unknown proteins

tryptic digestion products (peptide fragments)

A detection method

a computer database with known protein sizes

36
Q

Give a brief description of the purpose of conducting Western Blot, Centrifugation, Chromatography, and SDS page

A

Western Blot: amount/detection of a known protein

Centrigrucation: isolation of a protein(s)

Chromatography: purification of a protein(s)

SDS Page: separation of protein

Molecular 2018 (39 decks)

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