Exam 1: Ch 3 Protein Purification and Analysis

Question 1

overall task

Answer 1

to separate (resolve) a protein from others based on physical or chemical differences

Question 2

3 most widely used characteristics for separating proteins

Answer 2

size (length or mass)

net electrical charge

binding affinity for specific ligands

Question 3

mass

Answer 3

weight of a sample (in daltons or AMU) where density is the ratio of its mass to volume (g/L)

Question 4

proteins vary greatly in ____ but not ____

Answer 4

mass, density

Question 5

centrifugation

Answer 5

two particles in suspension with different masses or densities settle to the bottom of a tube at different rates

heavier or more dense molecules settle (sediment) faster than lighter or less dense molecules

Question 6

2 purposes centrifugation is used for

Answer 6

preparative technique to separate one type of material from another

analytical technique to measure physical properties of macromolecules

Question 7

differential centrifugation

Answer 7

separation of water-soluble proteins from insoluble cellular material

cell organelles and fragments form a pellet at the bottom of the tube, and proteins are in the supernatent

Question 8

rate-zonal centrifugation

Answer 8

separation of water-soluble proteins through a solution of increasing density called a density gradient (often sucrose)

proteins separate into bands/disks based on their mass

Question 9

electrophoresis

Answer 9

technique for separating molecules under an applied electric field

dissolved molecules move through an electric field at a speed determined by their charge to mass ratio

Question 10

SDS-PAGE

Answer 10

SDS ionic detergent denatures multimeric proteins

electrophoresis in polyacrylamide gel separates by size b/c of sieving action (small moves faster)

SDS binds proteins proportional to length of pp chain

Question 11

2D gel electrophoresis

Answer 11

combines SDS-PAGE with a pH gradient

1st separates proteins by charge then by mass

negative charge migrates toward anode, positive toward cathode until reach isoelectric point (pH when net charge of protein is 0)

Question 12

liquid chromatography

Answer 12

sample placed on a packed column of spherical beads in a cylinder and flows down via gravity or a pump

nature of the beads separate by mass, charge, or binding affinity

Question 13

gel filtration chromatography

Answer 13

separates proteins by mass

beads in columns have tunnels

small proteins get trapped in tunnels and move slowly, big proteins filter right through (more quickly)

Question 14

ion-exchange chromatography

Answer 14

separates proteins by charge

beads have carboxyl groups ( - ) or amino groups ( + )

negatively charged proteins get stuck to amino beads, and positive charged proteins get stuck to carboxyl beads

elute with high [ ] of salt solution

Question 15

affinity chromatography

Answer 15

ligands or mother molecules that bind to the protein are covalently attached to beads

antibody affinity, immunoaffinity are commonly used

Question 16

assay

Answer 16

detects presence of a molecule of interest as it is separated from other molecules

uses a distinctive characteristic of the protein like binding a specific ligand, catalyzing a specific rxn, or being recognized by a specific antibody

Question 17

chromogenic and light-emitting enzyme rxns

Answer 17

enzyme assays detect loss of substrate or formation of product

chromogenic substrates change color during the rxn

Question 18

antibody assays

Answer 18

antibodies specifically recognize the epitope of an antigen

presence of the epitope visualized by labeling antibody with an enzyme that is fluorescent or radioactive

green fluorescent protein found in jellyfish is used

Question 19

detecting proteins in gels

Answer 19

proteins embedded in 1 or 2D gels not usually visible

must label or stain proteins while they’re in the gel or electrophoretically transfer the proteins to a membrane of nitrocellulose and then detect them

proteins dyed with organic dye or silver based stain

Question 20

immunoblotting (Western blotting)

Answer 20

separate proteins and then identify a specific protein of interest by using 2 diff antibodies

1st antibody binds to epitope on target protein

2nd antibody binds to 1st antibody and has an enzyme or other molecule attached that generates color or light (chemiluminescence)

Question 21

epitope tagging

Answer 21

biosynthetically or chemically attaching epitope to an unrelated protein

antibodies generated by injecting intact protein or a fragment of the protein into a rabbit to induce antibody formation

Question 22

specific activity

Answer 22

amount of radioactivity per unit of material

measured in disintegrations per minute (dpm) per millimole

Question 23

half-life

Answer 23

time required for half the atoms to undergo radioactive decay

shorter half-life equals higher specific activity

Question 24

radioisotope

Answer 24

one atom in a radiolabeled molecule that is in a radioactive form

Question 25

common radioisotopes used

Answer 25

sulfur-35

phosphorus-32

iodine-125

Question 26

how is radioactivity detected

Answer 26

geiger counter: handheld

scintillation counter: mix with liquid containing fluorescent compound that emits a flash of light when it absorbs radiowaves

Question 27

pulse-chase experiment

Answer 27

trace changes in intracellular location of proteins over time

radiolabeled compound attached to cellular molecule of interest “pulse”

“chase” is incubation period when samples are taken to determine location of the pulse

Question 28

mass spectrometry

Answer 28

determine mass of a protein or fragments

ratio of mass of a charged molecule to its charge

m/z

Question 29

4 key features of a MS

Answer 29

ion source that transfers protons to the protein

mass analyzer in a vacuum separates ions of basis of different m/z

detector measures relative abundance of each ion in the sample

computer data system calibrates instrument

Question 30

tandem MS/MS

Answer 30

ions break protein into short peptides (less than 25 aa) at the peptide bonds

can determine aa sequence via deduction

Question 31

determine primary structure with gene sequences

Answer 31

MS performed to find mass fingerprint (list of MW of peptides from the protein)

MWs used to search a genome database to ID the sequences

Question 32

x-ray crystallography

Answer 32

beams of x-rays passed through a protein crystal to determine 3D structure

creates an electron density map that is analyzed and fit to a molecular model

Question 33

cryoelectron microscopy

Answer 33

protein sample frozen in liquid helium to preserve structure

then examined in frozen state by a cryoelectron microscope

Question 34

NMR spectroscopy

Answer 34

3D structure of proteins less than 200 aa studied this way

concentrated protein solution placed in magnetic field and effects of radio frequences on nuclear spin states of different atoms studied

determines distances between atoms