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Techniques Flashcards

1
Q

what is the first step in isolating cells?

A

disrupt the extracellular matrix and cell-cell junction

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2
Q

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

A

proteolytic enzymes and agitation

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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.

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4
Q

do cells usually circulate?

A

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

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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

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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.

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7
Q

what are antibodies?

A

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

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8
Q

do different B cells make a different antibody?

A

yes

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9
Q

how is amino acid sequence determined in antibodies?

A

it’s random

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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

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11
Q

what is a monoclonal antibody?

A

antibody secreted by a hybridoma cell line.

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12
Q

what is a hybridoma?

A

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

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13
Q

what do cells need to grow?

A

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

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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.

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15
Q

how is humidity controlled in those cell growy things?

A

water in a a low tray

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16
Q

how is pH controlled i those cell growy things?

A

CO2 is pumped in. sodium bicarbonate inside makes a buffer

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17
Q

what is the optimum temperature of cell growth?

A

37C

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18
Q

what is confluency?

A

too many cells growing on a petri dish

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19
Q

what is passaging or splitting cells?

A

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

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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

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21
Q

what are established cell lines?

A

immortalized cells, can grow forever in cell culture

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22
Q

what is HeLa?

A

isolated tumor cells from a women who died

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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
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24
Q

what are some ways to break open cells?

A

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

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25
what is osmotic shock?
cells "pop" due to osmotic pressure
26
what is centrifugation?
an instrument that rotates extracts of broken cells at high speeds
27
describe the common centrifuge.
the slots in the motor are at an angle and causes the pellet to sediment on the bottom sides
28
describe the swinging-bucket rotor.
the rotor has hinges and so when it spins, the samples are spun horizontally. pellet is strictly sediment ed on the bottom.
29
why does a centrifuge need a vacuum?
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
30
why does a centrifuge need to be balanced with samples?
itll hurt the poor rotor :(
31
how do you use a centrifuge to do organelle fractionation?
repeat the centrifugation at higher and higher speeds (whole cells, nuclei, cytoskeletons -> mitochondria, lysosomes, peroxisomes ->microsomes, small vesicles -> ribosomes, viruses, large macromolecules)
32
what is velocity sedimentation?
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
33
why is a sucrose gradient needed?
to prevent the bands from convective mixing, to keep each region denser than the one above it.
34
what is column chromatography?
a mixture of proteins in a solution is passed through a column containing a porous solid matrix
35
how are proteins separated by the matrix?
different proteins interact differently with the matrix and thus go through it at different rates
36
what is an ion-exchange chromatography?
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
what is a gel-filtration chromatography?
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
what is an affinity chromatography?
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
what is immunoprecipitiation?
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
what is a SDS-PAGE?
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
what is the role of SDS?
to break any protein-protein bonds so each protein molecule is reviewed separately. also coats the proteins with a negative charge
42
what is the role of beta-mercaptoethanol?
breaks any disulfide linkages in the proteins so individual proteins can be analyzed
43
what happens during an SDS-PAGE?
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
what is western blotting/immunoblotting?
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
how is mass spectrometry used to identify proteins?
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
what is co-immunoprecipitation?
the same as immunoprecipitation but you also look at what the protein is bonded with
47
once a binding partner is purified from co-ip or protein affinity chromatography then what?
western blot or mass spect
48
what is FRET?
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
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
co-ip and protein affinity unknown | FRET need
50
what is NMR?
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
what is cloning?
the act of making many identical copies
52
what is a plasmid vector?
small circular molecules of double-stranded DNA derived from plasmids that occur naturally in bacterial cells.
53
how are plasmids used as cloning vectors?
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
what is upstream?
towards 5'
55
what is downstream?
towards 3'
56
what are expression vectors?
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
what is the role of the promoter sequence in an expression vector?
causes unusually large amounts of mRNA to be produced from an adjacent protein-coding gene inserted into the vector
58
what are some methods for introducing membrane-impermeable substance into cells?
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
what is an epitope tag?
a fluorescent molecule
60
why do you need a tag?
need to differentiate between old and new proteins
61
what id GFP?
green fluorescence protein from jellyfish
62
how do you get a gene/cDNA from a protein?
determine AA sequence using mass spect, search DNA database for gene sequence, synthesize DNA primers for PCR, clone by PCR
63
how do you get a protein from a gene/cDNA?
insert protein-coding region of gene into expression vector, introduce into host cell to produce protein
64
what are genes/cDNA used for?
manipulation and introduce altered gene into cells/organism to study function
65
what are proteins used for?
determine 3-dimensional structure from x-ray or NMR, biochemical tests to determine activity
66
what is mutagenesis?
causing mutations
67
what is random mutagenesis
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
what is targeted mutagenesis?
you have the gene/protein and you want to study its function by changing parts of it and seeing what happens
69
how would you alter genes in a mouse?
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
how would you create a transgenic plant?
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
what is the cell doctrine?
all plants/animals are comprised of cells
72
what is the condenser?
focuses light on the sample
73
what is the tube lens
focuses image for eye
74
how does incident light work on a stained cell?
stained portion will absorb light of some wavelengths but allow other wavelengths to pass.
75
how does oblique incident light work on an unstained cell?
light scatters by components in the living cell can be collected to produce a bright image on a dark background
76
how does incident light work on an unstained cell?
generates contrast
77
what is bright-field microscopy?
light is transmitted straight through the specimen
78
what is phase-contrast microscopy?
phase alterations of light transmitted through the specimen are translated into brightness changes
79
what is differential-interferencontrast microscopy?
highlights edges where there is a steep change, looks 3D
80
what is dark-field microscopy?
specimen is lit from the side and only the scatter light is seen
81
how do you make tissue sections for slides?
1. stabilize proteins 2. freeze or embed in resin/wax 3. deli slice to create ribbons 4. stain
82
how are antibodies used as markers?
primary antibody attached to antigen then marker-coupled secondary antibodies attach to primary antibodies
83
how does a fluorescence microscope work?
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
why would you layer different fluorescent probes?
to see components of the cell at the same time
85
what is confocal microscopy used for?
a crisp image that is not blurred by the presence of fluorescent structures
86
how does confocal microscopy work?
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
how does multi-photon microscopy work?
fluorescent molecules get excited by multiple lower energy photons, can penetrate deeper
88
what is xray crystallography used for?
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)

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