Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

bio 340 > Techniques > Flashcards

Techniques Flashcards

You may prefer our related Brainscape-certified flashcards:
Biology 101 flashcards
1
Q

what is the first step in isolating cells?

A

disrupt the extracellular matrix and cell-cell junction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

how would you disrupt the extracellular matrix and cell-cell junction?

A

proteolytic enzymes and agitation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

how do proteolytic enzymes disrupt the extracellular matrix and cell-cell junction?

A

it digests proteins that bind on calcium, on which cell-cell adhesion depends on. then use mesh nets to grind into single cells.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

do cells usually circulate?

A

no, except for immune cells, they are part of tissue and are embedded in the extracellular matrix.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

what is a FACs and what is its function?

A

fluorescence-activated cell sorter. it helps separate a specific cell from a mixedcell suspension

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

how does a FACs work?

A

an antibody with fluoresent dye added for the specific cell wanted gets attached to the cell. the cells pass through a laser in a single stream and the fluorescence is measured. the cells are given a +/- charge depending on its fluorescence. then they are moved to their correct containers using a strong magnetic field.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

what are antibodies?

A

proteins made by B cells that bind with something, contain 2 heavy and 2 light chains

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

do different B cells make a different antibody?

A

yes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

how is amino acid sequence determined in antibodies?

A

it’s random

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

does the body keep all of its antibodies?

A

no, it deletes the ones that react to the body itself and keeps the ones that dont react

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what is a monoclonal antibody?

A

antibody secreted by a hybridoma cell line.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

what is a hybridoma?

A

Hybrid cell line generated by fusion of a tumor cell and another cell type. are immortal.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

what do cells need to grow?

A

attachment (something like extracellular matrix), nutrients, correct temperature, pH, and humidity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what are in nutrients for cell growth?

A

salt and pH balanced (usually colored to see if pH balanced). has calf serum that contains growth factors.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

how is humidity controlled in those cell growy things?

A

water in a a low tray

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

how is pH controlled i those cell growy things?

A

CO2 is pumped in. sodium bicarbonate inside makes a buffer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

what is the optimum temperature of cell growth?

A

37C

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

what is confluency?

A

too many cells growing on a petri dish

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

what is passaging or splitting cells?

A

take cells from a petri dish and transfer them onto another plate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

what are primary culture/cells?

A

cells taken from a live animal. as close to as real cells. won’t last too long bc when its outside the body it can’t get the right signals

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

what are established cell lines?

A

immortalized cells, can grow forever in cell culture

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

what is HeLa?

A

isolated tumor cells from a women who died

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

what are the steps of protein purification?

A
  1. break open the cells
  2. separation of sub-cellular fractions
  3. separation of proteins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

what are some ways to break open cells?

A

blender, ultrasound vibration, osmotic shock, forced through a small orifice ;)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Q

what is osmotic shock?

A

cells “pop” due to osmotic pressure

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
26
Q

what is centrifugation?

A

an instrument that rotates extracts of broken cells at high speeds

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
27
Q

describe the common centrifuge.

A

the slots in the motor are at an angle and causes the pellet to sediment on the bottom sides

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
28
Q

describe the swinging-bucket rotor.

A

the rotor has hinges and so when it spins, the samples are spun horizontally. pellet is strictly sediment ed on the bottom.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
29
Q

why does a centrifuge need a vacuum?

A

when the rotor spins it will hit the air molecules causing the instrument to heat up. if it heats up, the sample will also heat up and denature. the vacuum gets rid of the air molecules to reduce friction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
30
Q

why does a centrifuge need to be balanced with samples?

A

itll hurt the poor rotor :(

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
31
Q

how do you use a centrifuge to do organelle fractionation?

A

repeat the centrifugation at higher and higher speeds (whole cells, nuclei, cytoskeletons -> mitochondria, lysosomes, peroxisomes ->microsomes, small vesicles -> ribosomes, viruses, large macromolecules)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
32
Q

what is velocity sedimentation?

A

layering the sample in a thin band on top of a sucrose gradient, various components move down as distinct bands at different times. separates by size AND shape

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
33
Q

why is a sucrose gradient needed?

A

to prevent the bands from convective mixing, to keep each region denser than the one above it.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
34
Q

what is column chromatography?

A

a mixture of proteins in a solution is passed through a column containing a porous solid matrix

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
35
Q

how are proteins separated by the matrix?

A

different proteins interact differently with the matrix and thus go through it at different rates

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
36
Q

what is an ion-exchange chromatography?

A

the beads are charged and the proteins that stick on the beads are separated on whether they are positively or negatively charged. based on charge.

37
Q

what is a gel-filtration chromatography?

A

the beads have small channels that smaller molecules can get into but not the bigger ones. the smaller ones will elute slower. based on size

38
Q

what is an affinity chromatography?

A

beads are bonded with a specific substrate/antibody. proteins that can fit into that substrate can be eluted in a nearly pure form. can be eluted out with a concentrated substrate solution

39
Q

what is immunoprecipitiation?

A

beads with specific antibodies is added to the protein extract and mixed. the protein is allowed to bind to the antibody and then the solution is centrifuged, leaving the beads on the bottom. usually used in small amounts

40
Q

what is a SDS-PAGE?

A

an electric field is applied to a protein solution and it causes the protein to migrate through the gel that depends on its net charge, size, and shape

41
Q

what is the role of SDS?

A

to break any protein-protein bonds so each protein molecule is reviewed separately. also coats the proteins with a negative charge

42
Q

what is the role of beta-mercaptoethanol?

A

breaks any disulfide linkages in the proteins so individual proteins can be analyzed

43
Q

what happens during an SDS-PAGE?

A

when an electric field is applied to the gel, the negatively charged proteins will start traveling towards the anode (+) at different rates based on their size. big proteins will move slower bc of drag.

44
Q

what is western blotting/immunoblotting?

A

the gel is transferred onto a membrane with a strong electric current. it is then soaked into a specific antibody solution to reveal the protein of interest

45
Q

how is mass spectrometry used to identify proteins?

A

digested proteins are mixed with organic acids and dried onto a slide, a laser blasts a sample into ionized gas, ionized peptides accelerate in electric field and fly towards detector, how long it takes to get there depends on mass and charge. used with peptide database to identify protein

46
Q

what is co-immunoprecipitation?

A

the same as immunoprecipitation but you also look at what the protein is bonded with

47
Q

once a binding partner is purified from co-ip or protein affinity chromatography then what?

A

western blot or mass spect

48
Q

what is FRET?

A

2 proteins of interest are labeled with a different fluorescent protein, the emission of one is the absorption of the other. verifies if the two are binding partners

49
Q

of the following techniques, which ones do you have a have a potential binding partner and which ones can you find an unknown binding partner? co-ip, protein affinity choromatography, FRET

A

co-ip and protein affinity unknown

FRET need

50
Q

what is NMR?

A

requires a small volume of concentrated protein solution that is placed in a strong magnetic field. the hydrogen nuclei has a magnetic moment/spin. the spin can align or misalign when radio frequency is applied . when it returns to its normal state, RF is emitted depending on the environment of each hydrogen

51
Q

what is cloning?

A

the act of making many identical copies

52
Q

what is a plasmid vector?

A

small circular molecules of double-stranded DNA derived from plasmids that occur naturally in bacterial cells.

53
Q

how are plasmids used as cloning vectors?

A

the plasmid is cut with a restriction nuclease to create linear DNA molecules. the desired DNA id added to the plasmid and then covalently joined using DNA ligase. this recombinant DNA is put back into the bacterial cells that have been made to be permeable to DNA.

54
Q

what is upstream?

A

towards 5’

55
Q

what is downstream?

A

towards 3’

56
Q

what are expression vectors?

A

plasmids that have been designed to produce a large amount of stable mRNA that can be efficiently translated into protein when the plasmid is introduced to cells. easy to obtain proteins from choromatography

57
Q

what is the role of the promoter sequence in an expression vector?

A

causes unusually large amounts of mRNA to be produced from an adjacent protein-coding gene inserted into the vector

58
Q

what are some methods for introducing membrane-impermeable substance into cells?

A
  1. microinjection of the substance into the cel
  2. placing the cell between 2 electrodes and the substance. pores are temporarily open
  3. membrane-enclosed vesicles containing substance
  4. gold particles coated with DNA are shot into the cell
59
Q

what is an epitope tag?

A

a fluorescent molecule

60
Q

why do you need a tag?

A

need to differentiate between old and new proteins

61
Q

what id GFP?

A

green fluorescence protein from jellyfish

62
Q

how do you get a gene/cDNA from a protein?

A

determine AA sequence using mass spect, search DNA database for gene sequence, synthesize DNA primers for PCR, clone by PCR

63
Q

how do you get a protein from a gene/cDNA?

A

insert protein-coding region of gene into expression vector, introduce into host cell to produce protein

64
Q

what are genes/cDNA used for?

A

manipulation and introduce altered gene into cells/organism to study function

65
Q

what are proteins used for?

A

determine 3-dimensional structure from x-ray or NMR, biochemical tests to determine activity

66
Q

what is mutagenesis?

A

causing mutations

67
Q

what is random mutagenesis

A

randomly mutageneize organisms, find out which has the process affected. then you see what gene is involved then deduced which protein it is. you want to study a cellular process, no info

68
Q

what is targeted mutagenesis?

A

you have the gene/protein and you want to study its function by changing parts of it and seeing what happens

69
Q

how would you alter genes in a mouse?

A

alter a version of target gene and place into cells and let them form a colony. find cells that have the altered gene and place in mouse blastocyst. F2 will show results

70
Q

how would you create a transgenic plant?

A

punch holes in leaf and let them incubate with the desired gene, cells that have callus (undeveloped cells) growing will be used and a shoot-inducing medium is added then a root-inducing medium.

71
Q

what is the cell doctrine?

A

all plants/animals are comprised of cells

72
Q

what is the condenser?

A

focuses light on the sample

73
Q

what is the tube lens

A

focuses image for eye

74
Q

how does incident light work on a stained cell?

A

stained portion will absorb light of some wavelengths but allow other wavelengths to pass.

75
Q

how does oblique incident light work on an unstained cell?

A

light scatters by components in the living cell can be collected to produce a bright image on a dark background

76
Q

how does incident light work on an unstained cell?

A

generates contrast

77
Q

what is bright-field microscopy?

A

light is transmitted straight through the specimen

78
Q

what is phase-contrast microscopy?

A

phase alterations of light transmitted through the specimen are translated into brightness changes

79
Q

what is differential-interferencontrast microscopy?

A

highlights edges where there is a steep change, looks 3D

80
Q

what is dark-field microscopy?

A

specimen is lit from the side and only the scatter light is seen

81
Q

how do you make tissue sections for slides?

A
  1. stabilize proteins
  2. freeze or embed in resin/wax
  3. deli slice to create ribbons
  4. stain
82
Q

how are antibodies used as markers?

A

primary antibody attached to antigen then marker-coupled secondary antibodies attach to primary antibodies

83
Q

how does a fluorescence microscope work?

A

consists of 2 barrier filters that only lets certain wavelengths through. and a beam-splitting mirror that reflect or transmits wavelength based on length

84
Q

why would you layer different fluorescent probes?

A

to see components of the cell at the same time

85
Q

what is confocal microscopy used for?

A

a crisp image that is not blurred by the presence of fluorescent structures

86
Q

how does confocal microscopy work?

A

fluorescent specimen is lit up with focused light from a pin hole
emitted light from fluorescence is focused on the pinhole and reaches detector
emitted light from out-of-focus at pinhole is excluded from detector

87
Q

how does multi-photon microscopy work?

A

fluorescent molecules get excited by multiple lower energy photons, can penetrate deeper

88
Q

what is xray crystallography used for?

A

can determine protein structure. x-rays are directed at sample and a small fraction are scattered by atoms in the sample.

bio 340 (13 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions