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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
Q

describe what SDS does

A

denatures proteins

26
Q

the SDS unfolding does what to proteins (specifically)

A

catastrophic unfolding
structural disruption completely denatures polypeptides
separated all the chains of a multimeric protein into individiual
denatured polypeptides

27
Q

how does SDS denature proteins

A

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
Q

what does SDS PAGE stand for

A

sodium dodecylsulfate polyacrylamide gel electrophoresis

29
Q

describe SDS PAGE

A

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
Q

describe relationship between migration rate and protein size

A

migration rate is inversely related to protein size

31
Q

what can have an impact on protein mobility during SDS PAGE

A

post translational modifications

32
Q

describe a post translational modification that affects SDS PAGE

A

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
Q

what is isoelectric point

A

pI
pH at which sum of all charges = 0

34
Q

what does isoelectric point depend on

A

amino acid composition of each protein

35
Q

describe when pI is high or low

A

high if many basic residues
low if many acidic residues

36
Q

describe isoelectric focusing

A

pH gradient established using special buffers immobilized in acrylamide gel
proteins are subjected to electric field and migrate

37
Q

name the special buffers used in isoelectric focusing

A

ampholytes

38
Q

describe results of isoelectric focusing

A

proteins resolved in narrow stripes
each at its isoelectric point (pl)

39
Q

describe 2D gel electrophoresis

A

isoelectric focusing followed by SDS PAGE

40
Q

describe results pf 2D gel electrophoresis

A

no correlation between molecular weight and pI
separated protein spots are widely distributed - this reveals simultaneously many different proteins

41
Q

describe mass spectrometry

A

analytical and not preparative method
high precision determination of charge to mass ratio of ionized molecules

42
Q

describe the 3 concepts of mass spectrometry

A

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
Q

if a molecule carries a single charge then…

A

m/z=MW

44
Q

what is electrospray ionization

A

process for generating gas phase ionized molecules

45
Q

describe electrospray ionization

A

pump liquid in electrospray needle
ions released into atmosphere then goes to mass spec

46
Q

what happens next to gas phase ions generated by electrospray ionization

A

they are separated in mass analyzer into separate populations differing in m/z

47
Q

what is MS/MS (tandem MS)

A

recovering an ion
fragmenting it by high energy collision with an inert gas
doing mass spectroscopy on fragments

48
Q

describe MS/MS (tandem MS)

A

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
Q

Does the fragmentation in MS-MS break every peptide bond in every molecule, reducing the peptide to its individual amino acids?

A

NOOOO
fragmentation is partial and random

50
Q

what does partial fragmentation mean in MS-MS

A

only a small number or one of the peptide bonds per molecule is broken

51
Q

what can MS/MS identify

A

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
Q

what are proteomics

A

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
Q

can it fly?

A

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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