Protein IV

Question 1

dialysis

Answer 1

separates based on size

Question 2

dialysis elution

Answer 2

large molecules retained, small molecules diffuse out

Question 3

centrifugation

Answer 3

separates based on size and density

Question 4

centrifugation elution

Answer 4

larger, more dense proteins sediment faster

Question 5

size exclusion (gel filtration) chromatography

Answer 5

separates based on size

Question 6

size exclusion chromatography elution

Answer 6

large proteins elute first, small proteins elute last; small molecules get stuck in porous beads

Question 7

anion exchange chromatography

Answer 7

separates based on negative charge

Question 8

anion exchange elution

Answer 8

positively charged proteins will not bind and elute first; most negatively charged elute last

Question 9

pH < pKa

Answer 9

positive charge

Question 10

cation exchange chromatography

Answer 10

separates based on positive charge

Question 11

cation exchange elution

Answer 11

negatively charged proteins do not bind and elute first; the most positive will elute last

Question 12

affinity chromatography

Answer 12

separates based on specific interaction with protein

Question 13

affinity elution

Answer 13

only specific protein will bind to column

Question 14

HPLC

Answer 14

higher resolution, better separation

Question 15

gel electrophoresis

Answer 15

separates based on size

Question 16

gel electrophoresis elution

Answer 16

small DNA molecules run faster than large molecules

Question 17

SDS-PAGE

Answer 17

denatures protein for uniform charge; separates based on size

Question 18

SDS-PAGE elution

Answer 18

small proteins faster than large

Question 19

isoelectric focusing

Answer 19

separates based on pKa

Question 20

isoelectric focusing elution

Answer 20

proteins with highest pKa run closest to negative electrode; proteins with lowest pKa run closest to positive electrode

Question 21

western blot

Answer 21

detects specific proteins

Question 22

western blot info

Answer 22

proteins are separated by SDS-PAGE prior to blot

Question 23

ELISA

Answer 23

detects specific proteins

Question 24

ELISA info

Answer 24

proteins are not separated

Question 25

peptide fingerprinting

Answer 25

detects peptides from proteolytic digestion of protein

Question 26

peptide fingerprinting info

Answer 26

separates based on size in one direction and charge in the other

Question 27

edman degradation

Answer 27

detects amino acid sequence of protein

Question 28

mass spectrometry

Answer 28

detects molecular mass of protein; can compare to known proteins in database to find what the protein is

Question 29

NMR spectrometry

Answer 29

detects protein structure and dynamics

Question 30

x-ray crystallography

Answer 30

detects protein structure

Question 31

cryo-electron microscopy

Answer 31

detects structure of protein complexes

Question 32

dialysis in medicine

Answer 32

in renal failure, urea and other small solutes accumulate in the blood --> dialysis is used to remove the small solutes (they can cross dialysis membrane, but blood cells will stay put)

Question 33

the greater the sedimentation coefficient _

Answer 33

the faster particles will sediment

Question 34

centrifugation in blood

Answer 34

RBCs will sediment faster, resulting in red blood cells at bottom and plasma at top

Question 35

predicting elution in size exclusion

Answer 35

weight is determined by total MW (not just subunit) --> a 56.6 tetramer is actually 226.4

Question 36

anion exchange setup

Answer 36

column is loaded with positively charged anions to trap negative proteins; to displace these bound negative proteins and allow them to elute, Cl- is added

Question 37

cation exchange setup

Answer 37

column beads are negatively charged to trap positive proteins; Na+ is added to displace the bound positive proteins and allow them to elute

Question 38

pH < pI

Answer 38

net positive charge

Question 39

pH > pI

Answer 39

net negative charge

Question 40

isoelectric focusing in medicine

Answer 40

sickle cell hemoglobin has higher isoelectric point than regular

Question 41

western blotting setup

Answer 41

SDS-PAGE separates proteins --> transfer to nitrocellulose --> sheet incubated with an antibody that recognizes proteins of interest

Question 42

ELISA

Answer 42

similar to Western blotting but proteins are bound to a solid support

Question 43

direct ELISA

Answer 43

reporter enzyme is coupled directly to the primary antibody

Question 44

indirect ELISA

Answer 44

reporter enzyme is coupled to secondary antibody

Question 45

capture assay ELISA

Answer 45

an antibody is bound to the solid surface then the antigen is added --> to detect antigen a primary antibody is added --> excess antibody is washed off --> secondary antibody is added --> washed off --> color reaction

Question 46

polyclonal antibodies

Answer 46

mixture of several antibodies that are usually produced by different B cell clones of an animal

Question 47

monoclonal antibodies

Answer 47

antibodies generated from identical B cells that recognize a single epitope

Question 48

polyclonal antibody formation

Answer 48

inject antigen into an animal --> after immunization, polyclonal antibodies can be obtained directly from serum

Question 49

monoclonal antibody formation

Answer 49

B-lymphocytes are isolated from the animal's spleen and fused with a myeloma cell line --> immortal B-cell-myeloma-hybridomas --> grow continuously in culture producing antibodies

Question 50

in isoelectric focusing, as pH increases _

Answer 50

proteins with low pIs will stop moving; those with highest pI's will stop moving at highest pH