amino acid and protein seperation methods

Question 1

stationary phase

Answer 1

particles of a solid with specific properties that can hydrogen bond to polar amino acids (ex. silica gel)

Question 2

mobile phase

Answer 2

liquid solvent or buffer that flows past the particles and is non-polar

Question 3

Partition chromatography

Answer 3

• 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

Question 4

Thin layer chromatography

Answer 4

• 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

Question 5

relative mobility

Answer 5

distance travelled by solvent front / distance traveled by sample
- very polar have high RF
- non polar have low RF

Question 6

highest point reached by the solvent in TLC

Answer 6

solvent front

Question 7

column chromatography

Answer 7

• amino acid separation
• sample mix ABC is applied at top of column, buffer is added and carries sample mix to collection tubes

Question 8

elution volume

Answer 8

volume of buffer needed to move a compound through a column
- compounds can be identified by their elution volume

Question 9

how are amino acids detected

Answer 9

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

Question 10

ion exchange chromatography

Answer 10

• 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

Question 11

Elution in ion exchange chromatography

Answer 11

• 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

Question 12

More characteristics of ion exchange chromatography…

Answer 12

• 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

Question 13

Metal affinity chromatography

Answer 13

• 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

Question 14

Gel filtration or molecular exclusion chromatography

Answer 14

• 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

Question 15

measuring molar mass of proteins through gel filtration

Answer 15

• 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

Question 16

ultracentrifugation

Answer 16

• 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

Question 17

electrophoresis

Answer 17

• 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

Question 18

SDS-Polyacrylamide gel electrophoresis

Answer 18

• 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

Question 19

how does SDS-Polyacrylamide gel electrophoresis separate proteins

Answer 19

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

Question 20

Isoelectric focussing

Answer 20

• 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

Question 21

What is the isoelectric point

Answer 21

pH at which the net charge on a protein is 0
- when reached the protein stops moving

Question 22

Two-dimensional gels

Answer 22

combines isoelectric focusing and SDS electrophoresis

Question 23

Mass spectroscopy

Answer 23

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