amino acid and protein seperation methods Flashcards
stationary phase
particles of a solid with specific properties that can hydrogen bond to polar amino acids (ex. silica gel)
mobile phase
liquid solvent or buffer that flows past the particles and is non-polar
Partition chromatography
- amino acid separation
- polar amino acids spend more time bonded to the stationary phase (silica) and move slowly
- non-polar amino acids spend more time in solvent and move almost as fast as solvent
Thin layer chromatography
- amino acid identification
- silica gel is thinly spread on a plastic sheet
- samples are applied on lower edge then placed in solvent
- different components of sample
move with the solvent at different
rates
relative mobility
distance travelled by solvent front / distance traveled by sample
- very polar have high RF
- non polar have low RF
highest point reached by the solvent in TLC
solvent front
column chromatography
- amino acid separation
- sample mix ABC is applied at top of column, buffer is added and carries sample mix to collection tubes
elution volume
volume of buffer needed to move a compound through a column
- compounds can be identified by their elution volume
how are amino acids detected
ninhydrin: reacts with primary and secondary amines, gives purple colour but yellow colour to proline, intensity of colour determines amino acid
flourescamine: gives yellow fluorescence under uv light
ion exchange chromatography
- separates on the basis of charge
- uses charged resins as stationary phase
- Cation exchanger resins contain negative groups which bind cations
- anion exchanger resins contain positive groups which bind anions
Elution in ion exchange chromatography
- competition with high ion conc. (NaCl) displaces the amino acids from the resin
- changing the pH to alter the charge on the amino acid so it no longer binds to resin
More characteristics of ion exchange chromatography…
- size of net charge on molecule determines how tightly the amino acid binds
- high Na+ in elution buffer first displaces weakly bound amino acids, more Na+ = more tightly bound amino acids being displaced
Metal affinity chromatography
- protein seperation method
- cluster of His in a protein binds tightly to Ni2+ or Co2+
- column is made up of chelating resin containing Ni2+
- His tagged protein is eluted by adding imidazole to the buffer which outcompetes His-tagged and protein no longer binds
- high degree of purification in one step
Gel filtration or molecular exclusion chromatography
- protein separation method
- separation on basis of size
- contains beads of polymeric gel which has many water-filled pores
- small molecules enter pores and are delayed in movement down the column
- medium molecules can only enter some pores are delayed less
- large molecules are excluded and stay in buffer flowing around beads, move through column fast
measuring molar mass of proteins through gel filtration
- measure the elution volume of proteins with unknown molar mass
- elution volume is the log of the molar mass
- then measure elution volume of unknown protein and project back to the log mass axis
ultracentrifugation
- protein separation method
- protein sample placed in an ultracentrifuge spinning and producing a large g-force
- molecules sediment at a rate depending on size and shape
- sedimentation volume is used to calculate molar mass of a protein
electrophoresis
- protein separation based on movement of charged molecules in an electric field
- faster = more charge, smaller
- slower = friction, larger
- rate of movement depends on size, shape and charge
- carried out in a porous gel
SDS-Polyacrylamide gel electrophoresis
- causes protein molecules to extend (denature) and gives a uniform charge per unit size
- native charge is swamped out, overall -ve charge moves protein towards +ve electrode
- may be used to measure Mm of an unknown polypeptide
- if there are multiple subunits in a protein, they usually separate
how does SDS-Polyacrylamide gel electrophoresis separate proteins
separation based strictly on size
- small proteins fit though all the pores and move rapidly down gel
- larger molecules meander to find pores to fit through, move more slowly
Isoelectric focussing
- separation of proteins based on isoelectric point
- at high pH, deprotonated protein moves toward the + electrode
- as it passes through the gradient of decreasing pH, becomes protonated and -ve charge decreases
What is the isoelectric point
pH at which the net charge on a protein is 0
- when reached the protein stops moving
Two-dimensional gels
combines isoelectric focusing and SDS electrophoresis
Mass spectroscopy
a way of identifying proteins by vaporizing them with a laser beam
- particles travel toward the detecter and velocity is based on size (smaller = faster)
- time to flight yields measurement 5 decimals accurate
