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Research Approaches > Exam > Flashcards

Exam Flashcards

1
Q

Name 3 utilizations of antibody research.

A

Isolate/Quantify level of protein expression, Diagnose diseases, and localize specific proteins

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2
Q

B lymphocytes differentiate into ____ which produce great quantities of antibody.

A

plasma cells

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3
Q

IgG is an example of a variable region (T/F).

A

False. Constant!

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4
Q

What are polyclonal antibodies?

A

Antibodies which link different epitopes of the same antigen. Generated by different B cells. Most robust + least costly

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5
Q

Polyclonal antibodies are not used for therapies (T/F).

A

True

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6
Q

How are polyclonal antibodies produced?

A

In host animals after injecting them with antigen 4/5 times. Then antibodies are isolated from serum and purified in colony.

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7
Q

Monoclonal antibodies bind only one epitope (T/F)

A

True

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8
Q

How are monoclonal antibodies produced?

A

From a B cell clone called a hybridome. More costly but also more specific and less variable. Utilized in research + therapy

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9
Q

How are hybridomes formed?

A

Fuse an isolated B cell from an immune rat with a plasma cell (myelome). This cell can then produce antibody at a grand scale.

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10
Q

What can ELISA be used for?

A

Infection diagnosis and quantify concentration of different substances in the blood like hormones, cytokines, and chimiokines.

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11
Q

How do immunological tests work?

A

Well plates coated with specific “capture” antibodies which permit immobilisation. The plate is rinsed of everything but the specific antigen. The detection antibody conjugates with an added enzyme marker. The produced signal is proportional to quantity of antigen present.

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12
Q

What is the smallest amount of antigen that ELISA can detect?

A

10^-12 M

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13
Q

What is the ELISA sandwich?

A

Most specific and precise along with competitive. Relies on linkage of two antibodies for the same antigen.

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14
Q

What is competition ELISA like?

A

Competition between marked antigen and an unmarked recombinant protein. The signal spreads gradually up to where the non-marked protein takes the place of the marked one. Allows for precise measure of number of present antigens.

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15
Q

What is the difference between direct and indirect immunomicroscopy?

A

Direct: 1 antibody is used for detection.
Indirect: Secondary antibody is used for detection.

In both, samples can be frozen/fixed and need to be permealized.

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16
Q

How does electronic immunomicroscopy work?

A

Antibodies conjugates with microparticles of gold which don’t let electrons pass and show up black on the sample. Different sizes can be used to detect two antigens at once.

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17
Q

What is epitope mapping?

A

Process that permites localisation of an epitope. Crucial for development of monoclonal antibodies for therapies and vaccines.

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18
Q

How does overlapping peptide scan work?

A

Type of epitope mapping. Antibody is added to matrix with antigen peptides that overlap partially. Binding is detected from array signal.

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19
Q

What is flow cytometry (FACS)?

A

Technique where lasers count cells and measure protein expression by single particles. Measure of forward scatter detects sell size. Cell granularity and level of fluorescence emitted by a detection antibody is detected by 90 degree refraction.

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20
Q

What is flow cytometry for?

A

Permits analysis of multiple physical and molecular parameters of thousands of cells per second. Uses a sorter (trieur) to separate different populations present.

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21
Q

Why is microscopy usually done?

A

Evaluate integrity, visualize small details and biochemical events in living cells.

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22
Q

0.5 um is ____ and 0.2 um is ____.

A

standard (light microscope); STED (super-resolution electromicroscopy)

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23
Q

What is the magification of electromicroscopes and what do they look at?

A

200 000x / fixed specimens ONLY

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24
Q

What are animal cells frozen in for microscopy analysis?

A

Isopentane

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25
Q

When are fresh vs. fixed cells used for microscopy?

A

Fresh is for live cells for quick verification (blood, sperm, culture)
Fixed is to preserve localization of proteins through cross-linking. Uses formaldehyde, glutaraldehyde, ethanol, or methanol.

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26
Q

Name the steps of preparing a microscopy sample.

A

Dissection, Embedding, Sectioning, Staining, Image Acquisition, Analysis/Measurements, Data/Statistical Analysis, Study Report

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27
Q

How does sectioning work?

A

For tissues, uses a microtome or cryostat at -20 to 10-100 um. Lame en verre for optic microscopy and grille en cuivre for electronic.

For cells, no cutting. Directly fixed to lame en verre.

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28
Q

What is the best light source for light microscopy?

A

Halogen / DEL

Lampes au mercure, xenon, ou DEL/laser for fluorescence

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29
Q

What is the advantage of an inverted microscope?

A

Plus d’espace sur la platine pour manipuler les specimens. Allows for micro-injection tools.

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30
Q

Name the two types of lens applications (correction optique).

A

Fluorite (less corrective) and Plan Apochromat

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31
Q

What does numerical aperture measure?

A

Capacity of the objective to collect light from the specimen. Proportional with resolution.

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32
Q

Why is immersion oil/water used?

A

When the light emitted is very weak, like in fluorescence. Decreases refraction and raises spatial resolution.

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33
Q

Brightfield has high contrast compared to phase and DIC contrast (T/F).

A

False, it’s the opposite.

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34
Q

What is the most powerful and common method of optical contrast.

A

Fluorescence

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35
Q

Name 3 uses of fluorescent dyes.

A

Mark antibodies and proteins, measure ion concentration, and analyse functions like membrane potential

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36
Q

Describe Epi-Fluorescence.

A

All emitted fluorescence is captured at one time. Excitation filter lets only specific length of light wave through dichronic mirror. Mirror leads excited light to specimen and lets specific length of light waves back up. The whole specimen is fluorescent, image is much clearer with a thin sample.

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37
Q

How do confocal microscopes work?

A

Pinholes in front of fluorescent detector block out-of-focus light. Light source is a “faiseau laser”, 3-4 to cover light spectrum. CCD detectors like in telephones are cooled to limit electric noise. MUCH clearer image than normal epifluorescence

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38
Q

La microscopie confocale permet de faire de la ________ en prenant des photos en serie a des plans focaux differents.

A

tomographie

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39
Q

Describe laser scanning (LSCM) and multipoint spinning disk.

A

Focused laser scans and reconstitutes the specimen using the reflected light. First to be developed.

Perforated wheel goes on top of laser. Holes allow light to pass both ways from many parts of the cell simultaneously.

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40
Q

Compare spinning disk to laser scanning.

A

Spinning disk is faster so it lets us visualize rapid events in cells, less phototoxic, better for live cells as it doesn’t kill them.

41
Q

What is lightsheet microscopy?

A

Emits a thin slice of light (100 nm - 3 um) perpendicular to specimen, excellent resolution based on thickness of light. Contrary to LSCM which excites one point at a time, this collects a whole slice. 100-1000x more rapid and less phototoxic

42
Q

What are the 2 main types of electron microscopy?

A

TEM and SEM

43
Q

La longeur d’onde d’un electron et 100 000x plus _____ que les photons. ________ jusqu’a 1-50 000 000X.

A

courte; grossissement

44
Q

TEM produces a 2D image and is very similar structurally to light microscopes (T/F).

A

True. Both use a source+condenser, lens system, and camera.

45
Q

What are the main differences between TEM and light microscopy?

A

TEM light source uses a Tungsten filament where electrons are accelerated by an anode at high voltage (40-400 KeV) before focalizing specimen.
Lenses are magnets.
Magnification is adjusted by magnetic current of lenses.

46
Q

SEM functions on copper grid under a vacuum (T/F).

A

False. TEM.

47
Q

How are TEM samples prepared?

A

Chemically fixed and frozen / enrobed in resin. Ultrathin slices to let electrons though (60-90 nm versus 10-100 um). Heavy metal contrast like osmium, plomb, uranyl acetate.

48
Q

How is pseudo-3D done?

A

Specimen is slowly turned from +60 to -60. Photographed at each angle. Images are aligned and corrected to create a 3D reconstitution on computer. Different structures can be modeled this way. Can’t show volume because it’s still a very thin slice.

49
Q

Correlative microscopy facilitates ______.

A

High resolution TEM imaging of fluorescent microscopy elements.

50
Q

Name 3 methods of 3D electromicroscopy (diamond knife).

A

Array tomography (many slices + laborious), serial block face SEM (balayage microscope, scrape resin and image one at a time), and focused beam SEM (same as serial block but uses a laser to scrape instead of diamond)

51
Q

Protein concentration measurement requires which kind of chemical reaction?

A

Colour change

52
Q

Which gel is biomedical standard for electrophoresis?

A

Polyacrylamide (PAGE)

53
Q

How are polyacrylamide gels formed?

A

Liaison chimique entre Acrylamine (% changes porosity) et Bisacrylamide, initie par le persulphate d’ammonuim + TEMED. Coules a la verticale.

54
Q

Low acrylamide PAGE is for mass separation, high acrylamide (>10%) is for separation by protein charge (T/F).

A

False!

55
Q

What does SDS do?

A

Negatively charged detergent (sodium dodecyl sulfate) that linearises proteins proportionally by length. All proteins become negatively charged so movement is only affected by weight.

56
Q

What are “reductive conditions” with SDS-PAGE

A

Disulfide bonds are reduced which reduces covalent bonds and linearises proteins in a complex. Allows a more precise measure of MW.

57
Q

What is the purpose of comparing reductive and non-reductive sample?

A

Determine if the protein of interest is associated with other to assure normal function in vivo.

58
Q

What does coomassie (bleu-native) do?

A

Allows proteins to separate by weight only. Native-PAGE preserves protein-protein interactions and keeps functional form. Not linearised!!

59
Q

In PAGE 2D what happens between Native-PAGE / IEF-PAGE and SDS?

A

Lane is turned 90 degrees then placed at the top of SDS gel for a second migration once denatured.

60
Q

Point de focus isoelectrique (pl) est le pH auquel __________. Tres utile pour la _______ et ______ qui ne peuvent pas etre detectees par SDS.

A

la charge nette d’un proteine deviens egale a 0; glycosylation; phosphorylation

61
Q

What is the most sensitive protein dye?

A

Teinture d’argent

62
Q

Immunobuvardage permet de suivre des changements au cours du temps. L’autoradiographie permet de visualiser et quantifier des proteins dans un melange (T/F).

A

False! It’s the opposite.

63
Q

Sanger method involves labelling and cleavage of the ______.

A

N-terminal amino acid

64
Q

What is the new method of sequencing?

A

1D/2D PAGE. Cleave proteins with a trypsin. Use MALDI or ElectroSpray Ionization to ionize fragments before mass spectrometry. Mass is compared with peptide mass fingerprinting or de novo sequencing if it doesn’t show up in database.

65
Q

What two methods are used to determine tertiary structure?

A

rayon X crystalography and nuclear magnetic resonance

66
Q

Describe X-ray crystallography.

A

Specimen is bombarded with electrons in a sous vide chamber. Electrons are focused through microscope lens to obtain a high resolution image. Specimen pivots to different angles to make a 3D image.

67
Q

Name the steps of co-immunoprecipitation.

A

Prepare sample with monoclonal/polyclonal antibodies attached to agarose or metal beads. Then, protein complexes formed. Precipitated proteins are washed and eluted then analysed with SDS-PAGE. Can detect direct and indirect interactions.

68
Q

Which method allows us to see ephemeral in-vivo protein interactions and how?

A

BioID. Protein of interest is fused with biotin ligase BirA. In presence of biotin, it marks proteins that interact with protein of interest. Purification with streptavidin beads and then blot.

69
Q

Which type of protein detection increases intraceller calcium waves?

A

FRET. Cells change from green to red to indicate time and space resolved interaction between calmodulin and kinase over time.

70
Q

Which processes break down glucose, amino acids, and fatty acids, respectively?

A

Glycolysis, Trans/Deamination, and B-oxidation

71
Q

How many kcal are 1g of glucose, lipid, and amino acid?

A

4, 9, and 4.

72
Q

Dans la calometrie directe chez l’homme, la consommation de _____ est mesuree. Complex + costly.

A

O2

73
Q

Describe how indirect calorimetry functions?

A

Mask or mouthpiece (embout buccal) pour collecter les gas. O2 and CO2 levels in inhaled and exhaled air are measured with mass spectrometry. Ventilation measured with a debitmetre to find VO2 and VCO2 which allow to calculate oxidation of nutrients and energy production.

74
Q

What does respiratory quotient represent?

A

Oxygen requirement for metabolism. Glucose higher than lipid.

75
Q

Glucose metabolism produces more energy per liter of oxygen than lipid (T/F).

A

True

76
Q

Pourquoi la calorimetrie indirecte est un modele de boite noire?

A

Ca fournit pas d’info sur les voies metaboliques. On mesure simplement l’O2 qui entre est de CO2 qui sort.

77
Q

Isotopic dilution gives us the proportion of what?

A

Rate of appearance and disappearance of glucose

78
Q

Standard oxygraphy uses an electrode to measure level of _____ from ____.

A

O2; suspended cells.

79
Q

What are the drawbacks of standard oxygraphy?

A

Weak productivity, requires lots of cells, and only works on cells that can survive in agitated suspension.

80
Q

What does a Seahorse analysis sense?

A

Oxygen and pH from adherent cells.

81
Q

What do oligomycine, FCCP, and anticycin-A/rotenone. do?

A

Block ATP synthase to look at O2 consumption for ATP synthesis.
Decouple O2 and ATP synthesis to measure max aerobic capacity.
Block respiratory chain to measure O2 consumption separate from mitochondria.

Proton leak: mitochondrial respirations separate from ATP.

82
Q

Cytochrome C tests outer mitochondrial membrane integrity and antimycin A and rotenone have inhibitory effects (T/F).

A

True

83
Q

Which type of study has the best potential to explain links between genotype and phenotype?

A

Metabolomique, it changes the most quickly in response to cellular changes.

84
Q

La fluxomiqu combine les methodes de metabolomique aves des isotopes stables pour determiner le ______.

A

flux de metabolites dans une voie particuliere.

85
Q

What are the types of targeted metabolique?

A

Univariee/multivariee, supervisee ou non, et correlation/covariance.

Use cold organic solvents or cold grinding then a GC/RMN to measure relative abundance in m/z.

86
Q

What does non-targeted metabolics do that targeted does not?

A

Statistical and pathway analysis

87
Q

Name 3 reasons mice are used for experimentation?

A

Short gestational length with large litters.
88% homology with humans.
Easy to manipulate genome of embryonic stem cells.

88
Q

What are the 3 ways to introduce a transgene or knockout in mice?

A

Inject a retrovirus in 8 cell embryo stage.
Inject DNA into male pronucleus of fertilised oocyte.
Modify embryonic stem cells then inject into early embryo.

89
Q

Describe embryonic stem cell editing.

A

Electroporation, Injection, use PCR and southern blot to test recombination.
Use recombinated cells to inject into embryo then inject embryos into mice. Wait 3 weeks!

90
Q

Generation F1 has only 1 ______ whereas in F2, ___ will have both transgenes.

A

chimera allele; 1/4

91
Q

What is introduced in genes to cause knockout and how?

A

Neo^r
Introduce a vector containing it to ES from a trophoblast.
Select integrated cells with Neomycine (positive selection) and then Gancioclovir (negative; requires HSV-tk gene).

92
Q

CRISPR is like the _____ of bacteria.

A

adaptive immune system. the second time a bacteria is infected, CRISPR region is activated and Cas9 + ARNguide form a complex and cut it out.

93
Q

How does gene editing occur with CRISPR?

A

ARNguide is created to recognize a specific region like 18-20, which must be within 15pb of a PAM region. Once cut, sequence is repaired with non-homologous end joining which inserts a stop codon. Insertions can also be done.

94
Q

How is “knock-in” Crispr different from knockout?

A

Uses homologous recombination.

95
Q

Cre is a recombinant tyrosine derive from _____. LoxP sequences are _____ with an 8pb spacer between the two reflects. Cre/lox lets us inactivate specific genes after ____.

A

bacteriophage P1; palindromic; birth

96
Q

How do we express Cre only in specific tissues?

A

By adding tissue-specific promotors and crossing with “floxed” LoxP mice. F1 will be 50% heterozygous for Gene X. Cross with a LoxP homozygote. 1/4 will be knockout. 1/4 will be LoxP+ and express the gene of interest.

97
Q

Which controls are needed for Cre/Lox experiment?

A

homozygous loxP, cre transgene, and cre floxed heterozygous.

98
Q

How does CreERT2 work?

A

Expression of Cre is controlled by modified estrogen receptor. Only goes to nucleus in the presence of TAM or 4-OHT.

99
Q

What is the “knockout first” strategy?

A

Uses bacterial artificial chromosome in ES by homologous recombination. Gene trap targeting cassette. LacZ raporteur, cassette Neo, and loxP sites flank critical region. FRT sites recognized by Flp flank the lacZ and cassette Neo.

Cross with a Cre mouse without the exon to create a knockout with the LacZ tag. Cross with a Fllp mouse and then a Cre mouse to have a knockout with no tag.

Research Approaches (3 decks)

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