5 Flashcards

(108 cards)

1
Q

most important factor for deciding how to purify protein

A

cellular localization

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2
Q

why do you need to purify proteins

A

other molecules/ions/proteins may fuck with or modify ur protein, creating a heterogenous population

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3
Q

first step to understanding protein function

A

purification

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4
Q

what question do you ask yourself in order to know where to get your crude extract for purifying proteins

A

where am i getting the protein from? choose correct cell or tissue source is needed to get an adequate quantity and quality.

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5
Q

how can molecular biology help you get crude extract

A

genetically engineer protein of interest

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6
Q

intracellular protein crude extract generation extra step

A

lysing cell

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7
Q

physical methods of lysing cell

A

grinding, sonication, vortexing with glass beads

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8
Q

other non physical methods of lysing cell

A

osmotic pressure, chemical basis (detergents)

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9
Q

why is it bad to use osmotic pressure for lysing cells

A

interact with water, forming H bonds, denaturing protein (that’s why you need buffer, to preserve structure, when doing lysing)

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10
Q

how to get rid of the other shit in crude extract

A

centrigufation to separate supernatant from pellet of large organelles or insoluable. the bottom of the container will be pellet. this can also be used to isolate a particular organellle or cellular compartment if you know where it is

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11
Q

ultracentrifugation used to isolate what

A

used to isolate larger protein complexes

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12
Q

chromatography

A

differential particioning of molecule between mobile (buffer) and stationary (column) phase

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13
Q

protein absorbance

A

biomolecules absorb light at a specific wavelength

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14
Q

what measures protein absorbance

A

spectrophotometer

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15
Q

do most proteins absorb visible light

A

no

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16
Q

how does absorbance indicate concentration of protein in solution

A

absorbance at 280 nm from aromatic amino acids. the aromatic ones can absorb and relase light

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17
Q

how can stains be used to visualize or quantify proteins

A

coomassie blue can bind noncovalently, irreversibly for a bradford assay

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18
Q

beer lambert law common formula

A

molar extinction coefficient (depending on trp and tyr number) times concentration times path length of cuvette

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19
Q

beer lambert law log formula

A

log(light that went through/light transmitted)

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20
Q

size exclusion chromatography/gel filtration

A

column with resin of porous beads that allow small proteins to enter while large ones don’t go in. elongated may appear larger due to orientation

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21
Q

why do you need calibration with known MW proteins for size exclusion chromatography

A

because then you know the size of the unknown

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22
Q

void volume

A

size exclusion. anything larger than the fractional range of the column goes straight through.

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23
Q

elution volume

A

size exclusion. when the protein first starts to come out

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24
Q

total volume of column

A

size exclusion. Vt

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25
where do you want Ve (elution volume) for size exclusion chromatography
between V0 and Vt
26
ion exchange chromatography
separates AA or peptides based on net charge
27
CM ion exchange
cation exchange resin. bind positively charged peptides
28
DEAE (anion exchange) resin
attracts and binds negatively charged polypeptides
29
how are proteins eluted from the ion exchange chromatography
increasing salt concentration or changing pH
30
if something is at a pH higher than its pI, it is:
deprotonated and an anion
31
affinity chromatography (how does the resin work)
protein bind to column based on affinity for specific molecules or chemical groups. resin has covalently bound molecules or ligands that interacts with target with noncovalent interactions
32
how is bound protein passed in affinity chromatography
released by passing a solution with free molecules that compete for binding
33
pros of affinity chromatography
useful for concentrating proteins in small volume
34
Ni2+, NTA, and histidine
his when bound to NTA resin can bind to Ni2+, so 6-10 his residues can be added to proteins as a tag to help with purification
35
how to elute his tagged protein bound to Ni2+
imidazole. affinity for ni2+ or nta
36
fusion proteins in purification, how are they made
created by adding tags (stretches of amino acid introduced using recombinant dna)
37
how are fusion proteins used
affinity chromatography can isolate fusion proteins and conduct pull down assays based on non specific interactions
38
con of fusion proteins
may also have new functions
39
maltose binding protein does what
helps with solubility
40
where should you put a tag on a protein for purification
on the surface for accessibility, not on the active site to avoid messing with function
41
immunoprecipitation
form of affinity purification. POI is antigen, binds to Fab domain of antibody
42
interactions in immunoprecipitation
non-covalent interactions, complementary binding
43
precipitating out complexes in immunoprecipitation
complexes can be precipitated out by purifying antibody on beads
44
when can you use immunoprecipitation
if there are specific antibodies present for your POI
45
dialysis for protein purification (logic)
removes small molecules (salts, H+, ions, imidazole) that were used to elute proteins. diffusion allows small molecules to move from high concentration to low concentration
46
why do you need to do do dialysis
because it may fuck with experiments or assays
47
dialysis procedure
place sample in semi permeable dialysis bag and incubate with buffer. the bag would spin with stir bar
48
buffer exchange
basically changing the pH inside and outside of the dialysis bag. if the pH is higher outside the protons will come out, changing the pH of the stuff inside which may affect protonation state of side chains involved in chemical reactions or interactions
49
HPLC (high pressure liquid chromatography)
very fine beads and high pressure pumps to move sample through column achieving higher resolution of peaks
50
what resin is used in HPLC, what does it determine
usually silica covered in hydrocarbons. determines separation basis
51
reverse phase hplc is
when you separate based off of hydrophobicity. hydrophobic compounds interact stronger with column and have a longer retention time
52
for sds page, where are the largest proteins
least migrated, closest to the well
53
what is the most specific chromatography
affinity
54
SDS page full name
sodium dodecyl sulfate, polyacrylamide gel electrophoresis
55
what does SDS do to proteins
detergent, denatures protein, giving polypeptide uniform negative charge.
56
SDS to amino acid ratio
one SDS for every 2 amino acids
57
SDS giving uniform charge to proteins means what
same mass to charge ratio for polypeptide chains, migrate to anode
58
polyacrylamide gel creates what for SDS
mesh of cross-linked molecules that separate based on side
59
BME for SDS
reduce disulfide and separate quaternary structure
60
how to deduce size from SDS
comparing to MW markers
61
can you determine identity from SDS page
no
62
why do you need coomassie for SDS
because most proteins are colorless
63
immunoblotting
SDS first, transfer to polymer sheet (solid support membrane), block unbound regions with wash, add primary antibody and wash, add secondary antibody and wash, illuminate blot and measure fluorescence
64
what are unbound spots blocked with in immunoblotting (western blocking)
milk or BSA
65
how does the primary antibody work in immunoblotting
recognize protein of interest with linear sequence of amino acid
66
how does secondary antibody work in immunoblotting
specific to Fc of primary antibody, has a fluorescently labelled tag or enzyme
67
what does immunoblotting tell you
if protein of interest is present, approximate size based on where it is in the gel
68
tandem mass spectrometry can tell you what
can identify polypeptides after ionization and separation, can determine sequence and identify them based on mass to charge ratio, finding specific AA sequence. you know charge and mass of molecule, by comparing to known peptides
69
larger polypeptides in tandem mass spectrometry
may need to be cleaved
70
edman degradation
like dna sequencing for proteins. can be done for shorter fragments. removes amino acids starting from N terminus with a chemical reaction
71
mass spectrometry process
peptides bombarded with laser or high energy electron beam to create ionized fragments. products attracted to charged plate detector, analyzed in a mass analyzer by time of flight.
72
mass spec difficulties
some AAs have similar or the same mass
73
why is obtaining primary sequence from DNA sequencing not that useful
post translational modification
74
limitations of edman degradation
each amino acid needs to be purified and identified using column chromatography. 100 AAs max. N-terminal may be modified, affecting chemical rxn
75
what can be used to generate smaller fragments for edman
trypsin, chrmotrypsin, cyanogen bromide
76
trypsin
after lys and arg, only if pro is not in peptide bond.
77
chymotrypsin
bulky or hydrophobic chains (trp, tyr, phe, leu, met)
78
cyanogen bromide
after met
79
why do you need to know protein's physiological environment for designing an experiment for studying proteins
to mimic cellular conditions (temperature, pH, detergents, cofactors, substrates)
80
how can you study proteins experimentally
in vitro with purified proteins (test tube), or in fixed cells (in vivo) to determine structure, intracellular localization or binding partners
81
how to assays try to understand protein function in an experimental study
noncovalent interactions, chemical catalysis, and other biochemical properties to elucidate protein funciton
82
methods to study protein-protein interactions
co-immunoprecipitation and pull down assays, cross linking reagents, fret
83
methods to study protein-molecule interactions
transport assays, enzymatic reactions
84
why is it hard to study biomolecular interactions
noncovalent interactions may be transient and short lived
85
enzymes and fluorescent proteins that can be used to generate a visual response
luciferase, horse radish peroxidase, beta galactosidase based assays, bio ID
86
pull down assay. question asked, what is needed?
another form of affinity chromatography. does x interact with y? pull down x and see if y follows. need x specific antibody and y specific antibody.
87
experimental steps for pull down assays
immobilize x antibody on solid surface, break open cells, isolate beads, wash and detect bound proteins with western blot
88
what probe gel (i.e. which antibody) to use in western blot for pull down assay
Y not X because you already know X is there
89
chemical crosslinking
form covalent bonds between molecules, revealing inter and intra molecular organization of AA based on location
90
example of nonspecific chemical crosslinker
formaldehyde (glue everything)
91
what can you target directly with chemical cross linking
reactive side chains of AA: primary amines, carboxyls, carbonyls, suflhydryls
92
disulfide crosslinkers
occur between cysteines when there is oxygen or with cysteine modifying reagents
93
amine crosslinkers target
aldehydes, carboxylic acid
94
hydrazides target
aldehydes, ketones, carboxylic acids
95
how to know which functional groups to crosslink
number and location of functional groups. need to be on surface
96
how can chemical crosslinkers be used to assess proximity of binding partners
linkers of predetermined length can be used as spacers.
97
SDS page assessing interactions
under denaturation conditions can detect a change in molecular weight, covalent bond cross linking or separation of proteins
98
what else can assess separated proteins in complex
western blot and mass spectrometry
99
x ray crystallography stabilizing structures can be done with
chemical crosslinking
100
bio id function
proximity dependent biotinylation assay that can be used to identify protein-protein interactions
101
bir A
biotin protein ligase fused to a protein of interest, and will biotinylate close proteins (10nm)
102
how to purify and identify biotinylated proteins
streptavidin beads, identified with western blot or mass spec
103
microscopy benefits
proteins can be seen in real time and fixed environments
104
fluorescently tagged proteins tell you
where things are but does not confirm interaction
105
alternatives for fluorescently tagged proteins
fluorescent antibodies
106
fret and bret in general
microscopy based assays that detect distance dependent protein protein interactions
107
fret unique traits
needs emission of light from donor fluorophore (CFP) which excites nearby acceptor fluorophore (YFP). so if close enough, light will have a certain emission
108
bret unique traits
bioluminescence. doesn't need external light source. based on enzyme catalyzed reaction that produces light, acting as the donor. still releases light