lecture 6

Question 1

dialysis seperates proteins based on what?

Answer 1

size

Question 2

name charge based separation

Answer 2

ion exchange chromatography

Question 3

how does gel filtration separate proteins?

Answer 3

size

Question 4

how does ultracentrifugation separate proteins?

Answer 4

size

Question 5

what determines the purification behavior of proteins?

Answer 5

charged groups, hydrophobic regions, size, and solvation

Question 6

name the positively charged amino acids

Answer 6

lysine, histidine, and arginine

Question 7

name the negative charged amino acids

Answer 7

aspartate and glutamate

Question 8

what does sub-cellular fractionation allows?

Answer 8

yield, activity, and specific activity

Question 9

explain dialysis

Answer 9

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

Question 10

centrifugal concentrators are useful for what?

Answer 10

concentrating, desalting proteins

Question 11

explain gel filtration chromatography

Answer 11

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

Question 12

other name for gel filtration chromatography?

Answer 12

size exclusion chromatography

Question 13

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

Answer 13

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

Question 14

the larger column of size exclusion allowss for better what?

Answer 14

resolution

Question 15

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

Answer 15

smaller proteins

Question 16

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

Answer 16

inversely related

Question 17

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

Answer 17

.15

Question 18

what is volume of protein injected into size exclusion?

Question 19

ultracentrifugation allows you to do what? explain process

Answer 19

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

Question 20

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

Answer 20

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

Question 21

protein analysis by ultracentrifugation allows for what? (preparative)

Answer 21

sub cellular fractionation -

Question 22

protein analysis by ultracentrifugation allows for what? (analytical)

Answer 22

analysis of protein mass, density, shape, and bindind

Question 23

the sedimentation velocity of particle depends on what?

Answer 23

on mass, shape, density

Question 24

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

Answer 24

spheres would move faster

Question 25

would a more or less dense molecule move faster?

Answer 25

more

Question 26

when p

Answer 26

it sinks

Question 27

when p>1 what does moelcule do in ultracentrifugation?

Answer 27

it will float

Question 28

explain ion exchange chromatography

Answer 28

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

Question 29

in which process is injection volume not limited?

Answer 29

ion exchange chromatography

Question 30

elution order in ion exchange depends on what?

Answer 30

how tight the charge is on the beads

Question 31

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

Answer 31

negative and will be able to bind an anion exchange resin

Question 32

in ion exchange. if protein buffer is at ph

Answer 32

positive and will bind cation exchange resin

Question 33

explain cation exchange in ion exchange chromatography

Answer 33

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

Question 34

explain anion exchange in ion exchange

Answer 34

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

Question 35

explain affinity chromatopgraphy

Answer 35

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

Question 36

explain high performance liquid chromatography

Answer 36

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

Question 37

reverce HPLC useful for what?

Answer 37

sperating intact proteins for structural studies

Question 38

Sds causes what changes in gel electrophoresis?

Answer 38

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

Question 39

in gel electrophoresis do small or larger molecules move faster?

Answer 39

smaller moves faster

Question 40

gel elctrophoresis allows you to determine what?

Answer 40

protein purity, homogeneity, distrubution

Question 41

in gel elctrophoresis, migration is proportional to what?

Answer 41

mass

Question 42

relative mobility in gel electrophoresis is affected by which factors?

Answer 42

hydrophobicity, glycosylation, phosphorylation,

Question 43

does phosphorylation lower and raise the PI

Answer 43

lowers

Question 44

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

Answer 44

diffuse bands

Question 45

2D electrophoresis seperates proteins based on what?

Answer 45

based on PI

Question 46

name some advantages of 2D electrophoresis

Answer 46

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

Question 47

name disadvantages of 2D electrophoresis

Answer 47

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

Question 48

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

Answer 48

alpha amino group

Question 49

amino acid analysis answers which questions?

Answer 49

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

Question 50

name the steps of amino acid analysis

Answer 50

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

Question 51

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

Answer 51

amino acid analysis

Question 52

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

Answer 52

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

Question 53

how much protein do you need in amino acid analysis?

Answer 53

relatively large amount. 300pmol

Question 54

edman degredation removes amino acid from which terminus?

Answer 54

N

Question 55

edman is relatively straight forward interpretation and based on what

Answer 55

chromatographic retention time

Question 56

cyanogen bromide does what?

Answer 56

cleaves polypeptides on the carboxyl side of methionine

Question 57

does cyanogen bromide create smaller or larger peptides?

Answer 57

larger

Question 58

what does trypsin do?

Answer 58

hydrolyzes peptides on the carboxyl side of arginine and lysine residues

Question 59

LYs -c cleaves where?

Answer 59

C term of the lysine

Question 60

asp-n cleaves where

Answer 60

n term of asp

Question 61

chymotrypsin cleaves wher

Answer 61

tyr, trp, phe, leu, met and others

Question 62

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

Answer 62

phosphorylated

Question 63

mass spectrometry allows for what?

Answer 63

mapping proteolytic peptide masses

Question 64

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

Answer 64

small