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Biology Exam 2 > Analyzing Cells, Molecules & Systems > Flashcards

Analyzing Cells, Molecules & Systems Flashcards

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1
Q

cell culture

A

removal of cells from an organism, and promoting their subsequent growth in a favorable artificial environment

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2
Q

types of cell cultures

A
  1. primary cell culture

2. established or continuous cell lines

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3
Q

primary cell culture

A
  1. taken directly from animal
  2. involves enzymatic/mechanical selections steps to isolate cells from population
  3. cells survive for finite amount of time
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4
Q

examples of primary cell cultures

A

primary neurons

cardiomyocytes

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5
Q

established/continuous cell lines

A
  1. a primary culture that has been made immortal by transformation
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6
Q

most common established cell lines

A
  1. tumor derived

2. transformed via virus

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7
Q

example of continuous cell lines

A
  1. SH-SY-5Y — human neuroblastoma derived
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8
Q

cryopreservation

A

used to freeze cells

store in liquid nitrogen

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9
Q

advantages of cell culture

A
  1. study cell behavior w/out complexity that occur in animal
  2. cell characteristics can be maintained
  3. control growth for uniformity
  4. can expose to agents to see what happens (study drugs, chemicals, etc.)
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10
Q

sub-culture

A

growing cells and dividing into new

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11
Q

disadvantages of cell culture

A
  1. must develop techniques to maintain healthy cells
  2. quantity of material is limited
  3. dedifferentiation can occur - which impacts the biology of your cells – making them different than the original cells
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12
Q

applications of cell culture

A
  1. research
  2. disease simulation
  3. drug testing
  4. genetic analysis
  5. produce biological products (hormones, proteins)
  6. regenerative medicine
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13
Q

cell model of Parkinson’s disease

A
  1. expose SH-SY5Y cells to 6-OHDA
  2. Parkinson’s induced
  3. creates a reactive oxygen species
  4. apoptosis triggered
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14
Q

______ helps us to study the unique structure and _____ of individual proteins.

A

purification

structure and function

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15
Q

purification may use _______ to overexpress a protein - then purify it.

A

recombinant DNA technology

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16
Q

purification can target ______ proteins as well

A

endogenous - diseased

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17
Q

sub-cellular fractionation

A
  • -removes all other types of cells
  • -reduces the complexity of the material
  • -followed by purification
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18
Q

sub-cellular fractionation steps

A
  1. tissue sample - mechanical blending
  2. get homogenate
  3. centrifuge to separate cell types
  4. lysis of cells
  5. ultracentrifugation - separates organelles
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19
Q

homogenate

A

suspension of different cell types mixed

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20
Q

lysis of cells: sub-cellular fractionation

A

clean up the cells via
osmotic shock
ultrasonic vibration
mechanical blending

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21
Q

types of ultracentrifuge

A

fixed angle rotor

swinging bucket rotor–tubes are not rotating at a fixed angle

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22
Q

microsomes

A

when the ER breaks into little vesicles

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23
Q

when do you use the swinging bucket rotor?

A

for density gradient centrifugation

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24
Q

column chromatography

A

apply solvent continuously to top of column and collect fractionated molecules from bottom

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25
Q

different types of matrices for column chromatography

A
  1. ion-exchange chromatography
  2. gel-filtration chromatography
  3. affinity-chromatography
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26
Q

beads of column chromatography

A
  1. polar beads to attract a certain charge
  2. beads w/ specific pore size that can exclude larger particles
  3. beads that only attach to the protein desired
27
Q

techniques to analyze proteins

A
  1. SDS-PAGE
  2. western blot
  3. ELISA
  4. mass spectrometry
28
Q

SDS-PAGE

A

sodium dodecyl sulfate - polyacrylamide gel electrophoresis

used to analyze unknown proteins

29
Q

SDS-PAGE mechanism

A
  1. add sds to protein mixture
  2. entire protein is coated in sulfate - now neg. charged
  3. adds B-mercaptoethanol – reduces disulfides
30
Q

SDS

A

a largely hydrophobic negatively charged

unfolds proteins
gives all proteins a neg. charge

31
Q

what is the purpose of using SDS to give all proteins a negative charge?

A

allows for all proteins to migrate toward a positive charge in the presence of current

and sds unfolds proteins

32
Q

B-mercaptoethanol addition

A

reduces disulfides (S-S) into S-H and S-H

further denaturation of proteins

33
Q

separating proteins via SDS-PAGE

A

can apply stains/dyes to sample and can visualize separated proteins

separated out based on their size

34
Q

larger proteins move _____ than smaller proteins

A

faster

35
Q

western blotting

A

utilize immunoblotting to analyze specific/known proteins

36
Q

SDS-PAGE principle

A

separating proteins by size

37
Q

ELISA

A

enzyme-linked immunosorbent assay

like western blotting but in fluid

38
Q

ELISA principle

A

tests for the levels of a specific antigen or antibody concentrations in a sample

quantifies the number of antigens/antibodies

39
Q

measuring the amount of an antibody in a sample

A

indirect elisa

40
Q

measuring the amount of an antigen in a sample

A

sandwich elisa

41
Q

indirect elisa

A

measure amount of antibody

42
Q

sandwich elisa

A

measure amount of antigen

43
Q

types of ELISA

A

indirect or sandwich

44
Q

ex. of sandwich ELISA

A

pregnancy test

45
Q

identify unknown proteins

A

mass spectrometry

46
Q

mass spectrometry requirements

A
  • -requires tryptic digestion of products
  • -peptide fragments become charged
  • -results in a graph showing the mass of the proteins
  • -then can determine the peptide sequence and discover the protein
47
Q

SNPs

A

single nucleotide polymorphism

1 in 1,000 nucleotides differ between people

can be neutral, pathogenic or predisposing

48
Q

restriction endonucleases

A

cut DNA at specific sites

49
Q

analyzing DNA

A

agarose gel electrophoresis

–different than SDS-PAGE because DNA is already charged

50
Q

gluing DNA together

A

ligase reactions

easier w/ compatible ends

51
Q

matrix of DNA electrophoresis

A

agarose gel

**not same as electrophoresis for proteins

52
Q

genes can be cloned using _____

A

bacteria

53
Q

cDNA clones

A

DNA copy of mRNA

has no introns
much smaller than original gene
requires viral enzyme reverse transcriptase

54
Q

difference in DNA libraries

A

genes vs. gene products

55
Q

PCR

A

polymerase chain reaction

used to amplify isolated DNA region thousands of times

56
Q

you cannot perform PCR if you …… ?

A

don’t know the sequence of the gene of interest

57
Q

ex. application of PCR

A

diagnosing HIV

do pcr to replicate DNA and then run electrophoresis to find if particles exist

58
Q

quantitative PCR

A

qPCR
used to quantify copy number of a specific gene in two or more samples in real time

also uses fluorescence

59
Q

applications of qPCR

A
  • -detect levels of infectious agent

- -determine levels of gene expression

60
Q

RFLP

A

restriction fragment length polymorphism

detection of mutations
ex. sickle cell disease

61
Q

VNTR

A

variable number of tandem repeats
or short tandem repeats

useful in identification and severity of inherited diseases
ex. huntington disease

62
Q

uses of RFLP and VNTR

A

forensics

diagnostics – prenatal/newborn screening, id genetic carriers

63
Q

DNA microarrays

A

used to determine overall change in gene expression between 2 samples

ex. cancer vs. normal
young vs. old brain

Biology Exam 2 (9 decks)

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