cell bio > methods > Flashcards

methods Flashcards

(66 cards)

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1
Q

drives choice in experiment

A

hypothesis

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2
Q

determines what is measured and what is manipulated

A

variable

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3
Q

ensures that experiment is valid

A

control

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4
Q

most important factor of experimental design

A

hypothesis

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5
Q
  • what we change
  • time, concentration, dose
A

independent variable

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6
Q
  • what we measure
  • contraction, relaxation, secretion, mRNA levels
A

dependent variable

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7
Q
  • manipulation we are confident WILL give effect we are measuring
  • i.e. known drug
A

positive control

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8
Q
  • manipulation we are confident WILL NOT give effect we are measuring
  • i.e. saline
A

negative control

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9
Q

levels of analysis: organisms
- humans, animals

A

in vivo

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10
Q

levels of analysis: isolated tissue
- organ bath studies

A

ex vivo

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11
Q

levels of analysis: isolated cells
- cell lines

A

in vitro

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12
Q

simplest level of analysis

A

in vitro
- single cell type

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13
Q

which level of analysis would using engineering mice without a particular histamine receptor be?

A

in vivo

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14
Q

which level of analysis is difficult to interpret the results of? ethically/financially challenging to perform?

A

in vivo

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15
Q

experiments for ex vivo

A
  • ussing chamber
  • organ bath
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16
Q

experiments for in vivo

A
  • animals
  • humans
    (whole organism studies)
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17
Q

ussing chamber

A
  • membrane is placed in bath
  • on each side, there are diff. bathing medium chambers
  • can measure movement of molecules across membrane
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18
Q
  • whole organ or cut piece in bathing medium
  • measure contractions/other responses
A

organ bath

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18
Q

distinct advantage of ex vivo approaches

A
  • high degree of control over environment
  • responses measured can be relevant to organ function itself
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19
Q

pros/cons of ex vivo appraoch

A
  • pro: many cell types involved (cell=to-cell interactions expected)
  • cons: no neuronal/endocrine control
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20
Q

this level of analysis uses single cells or cell lines

A

in vitro

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21
Q

experiments for single cell

A
  • electrophysiology
  • genomic manipulation
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22
Q

experiments for cell lines

A
  • single isolated cell expanded/adapted to survive in dish/flask
  • can purchase with defined properties
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23
Q

which level of analysis gives exquisite control over environment/cell types involved

A

in vitro

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24
pros/cons of in vitro
- pro: exquisite control over environment and cell properties - con: difficult to generalize to more complex biological conditions, responses that can be measured aren't always obvious
25
two experimental approaches
- structural - functional
26
structural experimental approach
- measurement/assessment of structures - describes: properties, presence, location, sequence - who/what/where/how much/is it there?
27
functional experimental approach
- behaviour of the system - demonstrates: activities of structures, changes under different conditions, over time - when/how/what happens when - typically has time as variable
28
experimental experiments for structural approach
- microscopy - differential centrifugation - subcellular fractionation
29
experimental experiments for functional approach
- organ bath studies - cell lines - radio-isotopes/tracers - electrophysiology
30
types of microscopy
- light: light goes through sample, magnified by lenses before reaching eye/camera - electron: higher magnification, use electron beams, collimated (focussed) w/ electromagnets
31
types of electron microscopy
- SEM: show surface structure - TEM: show internal structures
32
differential centrifugation
- low speed: heavier things collect at bottom - use microscope to scan pellet contents at bottom
33
subcellular fractionation
- homegenize tissue (break them apart) - permeabilize them (w/ detergent) so contents are released but subcellular organelles are intact - sequentially centrifuge out diff organelles at diff speeds so each fraction has diff organelles
34
who created radio-isotope tracer methodology
georg hevesy
35
radio-isotopes and tracers
- if detectable, radioisotopes can be tagged and followed - fluorescent/chemiluminescent tags: add to cells/animals which can be genomically altered to express the tags
36
green fluorescent proteins
insert gene for a fluorescent protein into organism
37
electrophysiological techniques
look inside cells and how nerve impulses move along cells
38
who did the classical analysis of nerve function through an electrophysiological technique? how?
- study giant squid axon using extracellular electrodes - hodgkin and huxley
39
example of intracellular electrophysiological study? who did this?
- microelectrodes to study membrane potential btwn inside/outside of cell - ling and gerard
40
who invented patch clamp
- neher - sakmann
41
patch clamp
- put electrode on top of cell using suction - studied single channels - square electrode
42
molecular technology
- sequencing (dna/rna) - separation and identification: blots, ELISA, immunohistochemistry - PCR (polymerate chain rxn) - computational methods
43
most of what we have learned abt dna/rna is based on feature of _____
base complEmentation
44
important feature of proteins
they are very sticky
45
sequencing methods: determining primary structure
- DNA: chemical sequencing, chain termination method, dye-terminator sequencing, pyrosequencing - RNA: reverse transcribe to DNA, then sequence DNA - protein: mass spectrometry, edman degradation, mRNA prediction(dna, if no introns)
46
how do we selectively cleave 4 bases of DNA?
gel electrophoresis
47
edman degradation
- digest sample protein into smaller parts - cleave amino acids without disturbing other peptide bonds - labour intensive, but now automated - similar to sanger sequencing for dna
48
mass spectroscopy
- measures mass-to-charge ratio - computationally intensive for long chains - enzyme digest, vapourize, ionize, detect
49
what protein sequencing method do we simply send out a sample for?
mass spectroscopy
50
types of blots
- Southern blot: DNA - northern blot: RNA - western blot: protein - eastern blot: post-translational modifications - northwestern blot: rna-protein interactions - farwestern blot: protein-protein interactions
51
Southern blot
- take DNA sample cleaved w/ specific restriction enzymes and separate w/ gel electrophoresis - DNA fragments transferred to a membrane - membrane treated w/ DNA probe (radioactive or fluorescent tagged) to show if target DNA sequence is present
52
- intercalating agent used as fluorescent tag or nucleic acid stain for techniques like gel electrophoresis - when exposed to UV, fluoresces w/ 20x intense orange colour
ethidium brome = EtBr = bromoethane
53
types of antibodies
- monoclonal - polyclonal
54
polyclonal antibodies
- derived from cells that make antibodies - bind to many locations (epitopes) on original protein - specific to protein, not to one section of protein
55
monoclonal antibodies
- bind specifically to one epitope (location) on a single clone of cell tht makes antibodies -can be active site on receptor or allosteric site - v specific amino acid pattern
56
what are cells that make antibodies called?
B cells
57
antibodies derived from multiple B cells
polyclonal antibodies
58
antibodies derived from single clone of a B cell
monoclonal antibodies
59
selection medium for mammalian cell culture
HAT medium
60
to detect specific protein in a tissue sample with antibodies
immunohistochemistry
61
technique used to amplify small sequences of DNA
PCR
62
who developed PCR
kary mullis
63
most revolutionary technology in molecular biology
PCR
64
uses of PCR
- analyses tht need large amnts of DNA - detection of specific sequences - many modifications invented
65
computational analysis
- BLAST - nucleotide or peptide sequence search database - once we have sequence, plug in and see what it's similar to
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