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DMD biochem > lecture 6 > Flashcards

lecture 6 Flashcards

1
Q

dialysis seperates proteins based on what?

A

size

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2
Q

name charge based separation

A

ion exchange chromatography

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3
Q

how does gel filtration separate proteins?

A

size

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4
Q

how does ultracentrifugation separate proteins?

A

size

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5
Q

what determines the purification behavior of proteins?

A

charged groups, hydrophobic regions, size, and solvation

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6
Q

name the positively charged amino acids

A

lysine, histidine, and arginine

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7
Q

name the negative charged amino acids

A

aspartate and glutamate

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8
Q

what does sub-cellular fractionation allows?

A

yield, activity, and specific activity

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9
Q

explain dialysis

A

separates proteins based on size. larger proteins will stay in hte membrane

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10
Q

centrifugal concentrators are useful for what?

A

concentrating, desalting proteins

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11
Q

explain gel filtration chromatography

A

glass tbe contains carbohydrate beads, with small holes in them. smaller proteins will go into the beads and larger molecules will dilute first.

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12
Q

other name for gel filtration chromatography?

A

size exclusion chromatography

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13
Q

protein must be what in order for size exclusion to work? what happens if does not apply?

A

proteins must be freely soluble or they will stick to the beads and glass, not allowing them to pass through the bottom

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14
Q

the larger column of size exclusion allowss for better what?

A

resolution

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15
Q

during size exclusion, which size proteins experience higher mobile phase volume?

A

smaller proteins

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16
Q

in size exchange, elution volume is proporitional/inverse to molecular weight?

A

inversely related

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17
Q

how much salt necessary to prevent non specific interactions between proteins and beads?

A

.15

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18
Q

what is volume of protein injected into size exclusion?

A
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19
Q

ultracentrifugation allows you to do what? explain process

A

measure protein mass. form density gradient with sucrose, layer sample on top of gradient, place tube in rotor, proteins will stop when their density is equal to the solution

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20
Q

in ultracentrifugation, which size protein moves slowest? and what dictates this? (equation)

A

smaller molecules move. s value will tell you. smaller s means slower molecule and therefor the smallest molecule

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21
Q

protein analysis by ultracentrifugation allows for what? (preparative)

A

sub cellular fractionation -

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22
Q

protein analysis by ultracentrifugation allows for what? (analytical)

A

analysis of protein mass, density, shape, and bindind

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23
Q

the sedimentation velocity of particle depends on what?

A

on mass, shape, density

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24
Q

would spherical particles move faster or shorter than spherical ones during ultracentrifugation?

A

spheres would move faster

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25
Q

would a more or less dense molecule move faster?

A

more

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26
Q

when p

A

it sinks

27
Q

when p>1 what does moelcule do in ultracentrifugation?

A

it will float

28
Q

explain ion exchange chromatography

A

charged beads are in tube, opposite charges will stick, and others will discharge

29
Q

in which process is injection volume not limited?

A

ion exchange chromatography

30
Q

elution order in ion exchange depends on what?

A

how tight the charge is on the beads

31
Q

in ion exchange chromatography, if protein in buffer at ph> pi will protein be negative or positive?

A

negative and will be able to bind an anion exchange resin

32
Q

in ion exchange. if protein buffer is at ph

A

positive and will bind cation exchange resin

33
Q

explain cation exchange in ion exchange chromatography

A

positive charges proteins will bind to negative beads, and negative proteins will be released

34
Q

explain anion exchange in ion exchange

A

negative proteins will bind to positive beads, and positive beads will be released

35
Q

explain affinity chromatopgraphy

A

typical antibody purifictin. helps you zero in on what you really want

36
Q

explain high performance liquid chromatography

A

uses stainless steel, requires more pressure the bigger the molecule. involves a pumping system

37
Q

reverce HPLC useful for what?

A

sperating intact proteins for structural studies

38
Q

Sds causes what changes in gel electrophoresis?

A

causes all proteins to become negative, and they travel to the anode (positively charged end)

39
Q

in gel electrophoresis do small or larger molecules move faster?

A

smaller moves faster

40
Q

gel elctrophoresis allows you to determine what?

A

protein purity, homogeneity, distrubution

41
Q

in gel elctrophoresis, migration is proportional to what?

A

mass

42
Q

relative mobility in gel electrophoresis is affected by which factors?

A

hydrophobicity, glycosylation, phosphorylation,

43
Q

does phosphorylation lower and raise the PI

A

lowers

44
Q

mature protein in gel electrophoresis results in what type of band?

A

diffuse bands

45
Q

2D electrophoresis seperates proteins based on what?

A

based on PI

46
Q

name some advantages of 2D electrophoresis

A

separation and visualization of 1000s of proteins in 2 dimensions based on PI and molecular wight. easily visualized, comparison of protein expression as function of biological change, and many systems are available

47
Q

name disadvantages of 2D electrophoresis

A

usually only views proteins that are in large numbers. limited protein loading. proteins must be cut out in order to use the edman process. most animal proteins are modified and are observed in multiple spots

48
Q

in amino acid analysis, fluorescamine reacts with the _______ group of an amino acid to form a fluorescent derivative

A

alpha amino group

49
Q

amino acid analysis answers which questions?

A

is the protein pure, is it the right protein, does it have the expected amino acid composition?

50
Q

name the steps of amino acid analysis

A

hydrolyze protein into small amino acids, label amino acids with UV absorbing or fluorescent marker, separate different proteins with chromatography, measure the amounts of the amino acids based on intensity of marker associated with emergence of each type of amino acid

51
Q

which method is useful for comparing a protein to its theoretical sequence?

A

amino acid analysis

52
Q

amino acid analysis is used for what type of products? name the 3 examples

A

recombinant protein based products. recombinant drugs, antibodies, and animal feeds and additives

53
Q

how much protein do you need in amino acid analysis?

A

relatively large amount. 300pmol

54
Q

edman degredation removes amino acid from which terminus?

A

N

55
Q

edman is relatively straight forward interpretation and based on what

A

chromatographic retention time

56
Q

cyanogen bromide does what?

A

cleaves polypeptides on the carboxyl side of methionine

57
Q

does cyanogen bromide create smaller or larger peptides?

A

larger

58
Q

what does trypsin do?

A

hydrolyzes peptides on the carboxyl side of arginine and lysine residues

59
Q

LYs -c cleaves where?

A

C term of the lysine

60
Q

asp-n cleaves where

A

n term of asp

61
Q

chymotrypsin cleaves wher

A

tyr, trp, phe, leu, met and others

62
Q

if a protein has a higher mass, then it probably has been what?

A

phosphorylated

63
Q

mass spectrometry allows for what?

A

mapping proteolytic peptide masses

64
Q

in mass spectrometry, does small or large proteins come out first?

A

small

DMD biochem (7 decks)

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