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BIOL 200 > KH6 > Flashcards

KH6 Flashcards

1
Q

name the 4 physical and chemical properties that proteins can be analyzed by

A

mass or size (and shape)
density
electrical charge
binding affinity (for different ligands)

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2
Q

name the 3 separation methods used in biology

A

centrifugation
electrophoresis
chromatography

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3
Q

what is centrifugation

A

rapid psinning of the centrifuge tuble that generates a centrifugal force that is measured in g (unit of earths gravity, centrifuge can get up to 1000g)

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4
Q

describe what happens during centrifugation

A

force acts on particles (down to molecular size) suspended within the liquid medium of the centrifuge tube (usually aqueous)

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5
Q

name and explain the 2 type of centrifugation

A

swinging rotor bucket - tube swings up
fixed angle rotor - tube is fitted into a hole - so test tube cannot move - contents will run down tube and move to bottom

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6
Q

what happens when particles are denser than suspending medium (centrifugation)

A

g force will push particles to bottom of tube

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7
Q

what happens when particles are lighter than suspending medium (centrifugation)

A

g force will cause particles to float towards top of tube

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8
Q

what happens when particles are same density than suspending medium (centrifugation)

A

they will not move in any direction
they just stay where they are and diffuse around

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9
Q

usually are the particles we are interested in denser or lighter than the suspending medium (centrifugation)

A

usually they are denser than suspending medium
causes them to move to bottom of tube and forms pellet

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10
Q

what is rate of clearing supernatant of particles dependent on

A

size/mass of the particle (for particles of similar shape)
size unit calculated this way = the svedburg (S) - past ex: 40S ribosomal subunit, 28S rRNA

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11
Q

describe an example of differential centrifugation

A

separation of cellular contents by particle size/mass
mass of nuclei is larger than mitochondrion
low speed centrifugation pellets nuclei and leaves mitos in supernatant, so, must use higher speed centrifugation to pellet the mitos

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12
Q

describe the main aspects (the first few spins) of purification of coronavirus

A

infected cell homogenate is smashed up
first spin - medium spped, pellet nuclei and mitochondria
second spin - high speed to pellet virus (and everything that is similar size)

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13
Q

what do we have to do to virus particles in pellet (purification of coronavirus)

A

resuspend pellet
apply equilibrium density gradient centrifugation

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14
Q

what is goal of equilibrium density gradient centrifugation of purification of coronavirus

A

all of the particles in pellet are not all virus - way to separate
densities vary so can separate this way

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15
Q

describe equilibrium density gradient centrifugation

A

create a density gradient by smoothly mixing high and low density sucrose solutions while filling centrifuge tube
want denser sucrose at bottom and lighter at top

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16
Q

where do we apply the resuspended virus pellet in equilibrium density gradient centrifugation

A

top of sucrose gradient
then centrifuge at high speed

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17
Q

describe results of equilibrium density gradient centrifugation of coronavirus

A

virus will move toward bottom of tube but when it hits a solution of equal density it will stop moving down
density = 1.18g/mL
take fractions of the test tube and use sds page to visualize
covid is concentrated in fractions 5 and 6 - this was expected

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18
Q

what does electrophoresis depend on

A

charge mass ratio

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19
Q

describe electrophoresis

A

electric field generated by inserting electrode into liquid

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20
Q

for electrophoresis what is the direction and speed of migration determined by

A

direction = net charge
speed = net charge/mass ratio

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21
Q

describe what influences the rate of movement/direction in electrophoresis

A

if + then moves towards negative and vise versa
if -3 then it will move faster than just - since more charge

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22
Q

for electrophoresis what does + and - mean

A

+ = shows presence of basic amino acids side chains
- = shows presence of acidic amino acid side chains

23
Q

in gel electrophoresis what can happen

A

the migration of molecules may be impeded by the gel
larger molecules are impeded more - move slower

24
Q

what is a big breakthrough in protein electrophoresis

A

SDS
anionic detergent sodium docedyl sulfate
developed in 1960s

25
describe what SDS does
denatures proteins
26
the SDS unfolding does what to proteins (specifically)
catastrophic unfolding structural disruption completely denatures polypeptides separated all the chains of a multimeric protein into individiual denatured polypeptides
27
how does SDS denature proteins
interaction of its hydrophobic tail with hydrophobic aa side chains SDS hydrophobic tails bind to hydrophobic residues and itself - coats polypeptide in uniform layer SDS molecules are negatively charged to various parts of the SDS polypeptide chain repel each other and further disrupts and unfolds protein
28
what does SDS PAGE stand for
sodium dodecylsulfate polyacrylamide gel electrophoresis
29
describe SDS PAGE
SDS - denatures and makes all proteins negatively charged with the same charge:mass ratio SDS protein complexes put in gel matrix which impedes the larger molecules
30
describe relationship between migration rate and protein size
migration rate is inversely related to protein size
31
what can have an impact on protein mobility during SDS PAGE
post translational modifications
32
describe a post translational modification that affects SDS PAGE
phosphorylation of protein kinases can shift mobility of protein (and the apparent molecular weight) phosphate group interferes with SDS binding - so less SDS molecules and less motive force pushing it through (so results indicate protein is heavier than irl)
33
what is isoelectric point
pI pH at which sum of all charges = 0
34
what does isoelectric point depend on
amino acid composition of each protein
35
describe when pI is high or low
high if many basic residues low if many acidic residues
36
describe isoelectric focusing
pH gradient established using special buffers immobilized in acrylamide gel proteins are subjected to electric field and migrate
37
name the special buffers used in isoelectric focusing
ampholytes
38
describe results of isoelectric focusing
proteins resolved in narrow stripes each at its isoelectric point (pl)
39
describe 2D gel electrophoresis
isoelectric focusing followed by SDS PAGE
40
describe results pf 2D gel electrophoresis
no correlation between molecular weight and pI separated protein spots are widely distributed - this reveals simultaneously many different proteins
41
describe mass spectrometry
analytical and not preparative method high precision determination of charge to mass ratio of ionized molecules
42
describe the 3 concepts of mass spectrometry
1 - produce dispersed ions in a gas phase 2 - measure acceleration of the ions in an electric or magnetic field 3 - acceleration depends on mass/charge ratio (m/z)
43
if a molecule carries a single charge then...
m/z=MW
44
what is electrospray ionization
process for generating gas phase ionized molecules
45
describe electrospray ionization
pump liquid in electrospray needle ions released into atmosphere then goes to mass spec
46
what happens next to gas phase ions generated by electrospray ionization
they are separated in mass analyzer into separate populations differing in m/z
47
what is MS/MS (tandem MS)
recovering an ion fragmenting it by high energy collision with an inert gas doing mass spectroscopy on fragments
48
describe MS/MS (tandem MS)
take one of those peptides accelerate those ions in an electric field at very high velocity run them into inert gas like argon then energy and velocity is high that impact will break peptide bonds
49
Does the fragmentation in MS-MS break every peptide bond in every molecule, reducing the peptide to its individual amino acids?
NOOOO fragmentation is partial and random
50
what does partial fragmentation mean in MS-MS
only a small number or one of the peptide bonds per molecule is broken
51
what can MS/MS identify
second dimension of info provided by product ion spectrum analyzed computationally with respect to known protein sequences (based on computer translation of genome DNA sequences) can identify aa sequence of peptide ion
52
what are proteomics
analysis of biological protein samples by mass spectrometry and bioinformatics (computer analysis of DNA and protein sequences) in order to identify the population of proteins present in ant given subcellular organelle
53
can it fly?
can it be ionized - if it can then you can use mass spec to analyze it - need free ions floating

BIOL 200 (29 decks)

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