analytic techniques Flashcards
preparative purifications
produce significant quantities of purified product for ise
analytical purifications
smaller amount of a protein to identify, quantify
protein purification
to extract proteins by lysing the cell
protease inhibitors
prevent proteolytic degredation
centrifugation
separate particles based on mass and density
salting in
increase protein solubility
salting out
decrease protein solubility
- protein parcipitates out
mobile phase
carries mixture through a solid stationary phase where is interacts with the desired product material
stationary phase
solid
paper chromatography
stationary: filter paper
mobile: liquid solvent that carries solutes up the filter paper via capillary action
thin layer chromatograph
stationary: glass or plastic coated in absorbant silica (polar)
mobile: non polar liquid
- seperates molecules via polarity
advantage of TLC over paper chromatography
faster, more precise, versatile
retention factor (Rf)
how far a solute moves up the stationary plate
-approximates polarity
distance traveled through stationary plate / max distance traveled through the mobile phase
Rf range
between 0 -1
lower Rf
associated with more polar compounds
higher rf
associated with more non polar compounds
disadvantage of TLC
analytical purification only (doesnt purify )
column chromatography
stationary phase: stop cock that allows product to flow out
mobile: nonpolar
sequential flow between different columns to purify product
what elutes first in column chromatogrphy
protein of interest for isolateion
high performance liquid chromatography
high pressure
stationary phase: absorbant material with high pressure
mobile phase: liquid
- faster and more precise seperation of compounds
normal phase liquid chromatography
gas chromatography
mobile phase: gas
stationary phase: liquid
- measure volitile compounds
- sample is vaporized and travel at different rates due to polarity and boiling point
what happens to compounds with low boiling points in gas chromatography
vaporize first and elute first
size exclusion chromatography
column packed with gel beads and larger particles elute first
ion-exchange chromatography
selects for molecules with specific charge
- anion exchange and cation exchange
anion exchange chromatography
named after the type of ion the column is designed to attract
- anion sticks to stationary phase
- cations are eluted
- the column is covered in positive cahrges
cation exchange chromatography
positive ions stay in the tube
negative ions are eluted
tube is covered in negative charges
what is the column of an anion-exchange coated with
positive charges to attract the anions
what is the column of a cation-exchange coated with
negative charges to attract cations
what is eluted in cation exchange
anions
what is eluted in anion exchange chromatography
cations
affinity chromatography
binding affinity of proteins for specific ligands
stationary phase: contains ligand so sample passes through unwanted proteins
bond formation of affinity chromatography
noncovalent
immunoaffinity chromatography
antibody within the column to recover proteins bound to a specific antibody
electrophoresis
seperate molecules based on their migration in the electric field
gel electrophoresis
samples loaded onto agarose gel and electric field causes induction of a positive and negative pole
anode
positive pole
cathode
negative pole
electrolytic cells
current is applied to drive an otherwise nonspontaneous reaction
ex: gel electrophoresis
cathode is negative and anode is positive
galvanic cell
anode is negative and cathode is positive
- spontaneous already
criteria for distance traveled on gel electrophroesis
- charge
- size
SDS-PAGE
anionic detergent that gives proteins on a gel electrophoresis UNIFORM negative CHARGE so that proteins seperate only based on SIZE
what bonds does SDS disrupt
- non covalent bonds
- imparts even distribution of neg charge
- denatures
- does not efefct covalent bonds (disulfide bridges)
what bonds can SDS not disrupt
disulfide bridges
BME
disrupts disulfide bridges in sds page
what size proteins migrate more quickly in SDS page
smaller
what are proteins typically stained with in SDS page
comassie blue for visualization on the gel
isoelectric focusing
allows for fine seperation of proteins with different charge states using a pH gradient
- a protein in pH below the pI will migrate down the gel until it reaches its isoelectric point and will stop in the gel
spectroscopy
quantify amount of protein in a sample based on absorbance of light at a wavelength
bert- lambert law
A= Ecl
- a is absorbance
- e is constant
c is concentration of solute
l is path length in cm of transmitted light
what does a higher absorbance level mean
more protein is present
what wavelength do proteins absorb the most
uv range from 200nm to 400 nm
what wavelength do aromatic proteins absorb best
around 280 nm
what stain binds to all proteins for visualization
comassie blue
radioactive labeling
visualize proteins in an xray image
primary antibody
antibody that binds specifically to a protein
secondary antibody
binds to primary antibody and is tagged for visual bands in protein of interest in western blotting
what antibody is tagged in the western blotting
secondary antibody
radioimmunoassay
concentration of protein in sample is assesed by measuring extent to which unlabled proteins compete with radioactive labeled antigens for antibody binding sites
ELISA (enzyme linked immunosorbent assay)
antigens of a sample are attatched to a solid surface normally on a plate
antibodies specific to the antigen of interest are applied to the plate and these bind their protein antigens
- enzyme is covalently linked to an antibody and the enzyme substate is added to the reaction chamber
- enzyme catalyzes color change that can be measured to determine the amount of protein antigen present
western blotting
using antibodues specific to a protein of interest to be visualized after electrophoresis
what is the goal of elisa
to quanitfy the amount of antigen via visualizing an enzyme attatched to an antibody of the antigen
