Exam 1: Ch 3 Protein Purification and Analysis Flashcards
overall task
to separate (resolve) a protein from others based on physical or chemical differences
3 most widely used characteristics for separating proteins
size (length or mass)
net electrical charge
binding affinity for specific ligands
mass
weight of a sample (in daltons or AMU) where density is the ratio of its mass to volume (g/L)
proteins vary greatly in ____ but not ____
mass, density
centrifugation
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
2 purposes centrifugation is used for
preparative technique to separate one type of material from another
analytical technique to measure physical properties of macromolecules
differential centrifugation
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
rate-zonal centrifugation
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
electrophoresis
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
SDS-PAGE
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
2D gel electrophoresis
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)
liquid chromatography
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
gel filtration chromatography
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)
ion-exchange chromatography
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
affinity chromatography
ligands or mother molecules that bind to the protein are covalently attached to beads
antibody affinity, immunoaffinity are commonly used
assay
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
chromogenic and light-emitting enzyme rxns
enzyme assays detect loss of substrate or formation of product
chromogenic substrates change color during the rxn
antibody assays
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
detecting proteins in gels
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
immunoblotting (Western blotting)
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)
epitope tagging
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
specific activity
amount of radioactivity per unit of material
measured in disintegrations per minute (dpm) per millimole
half-life
time required for half the atoms to undergo radioactive decay
shorter half-life equals higher specific activity
radioisotope
one atom in a radiolabeled molecule that is in a radioactive form
common radioisotopes used
sulfur-35
phosphorus-32
iodine-125
how is radioactivity detected
geiger counter: handheld
scintillation counter: mix with liquid containing fluorescent compound that emits a flash of light when it absorbs radiowaves
pulse-chase experiment
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
mass spectrometry
determine mass of a protein or fragments
ratio of mass of a charged molecule to its charge
m/z
4 key features of a MS
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
tandem MS/MS
ions break protein into short peptides (less than 25 aa) at the peptide bonds
can determine aa sequence via deduction
determine primary structure with gene sequences
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
x-ray crystallography
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
cryoelectron microscopy
protein sample frozen in liquid helium to preserve structure
then examined in frozen state by a cryoelectron microscope
NMR spectroscopy
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
