lecture 5 - purification by chromatography Flashcards
1
Q
types of chromatography (4)
A
(1) charge - ion exchange
(2) polarity - hydrophobic
(3) affinity - binding specific ligand
(4) size
2
Q
ion exchange
- what is the solid phase
- what binds to it
- what elutes
- how to elute binded protein
A
- solid phase has a negative or positive charge
- proteins with opp charge to solid phase elute slowly
- proteins with same charge as solid phase elute faster
- elute protein by increasing [salt] or by changing the pH
3
Q
2 types of ion exchange chromatography
A
- cation exchnage = pos ions in solid phase
- anion exchange = neg ions in solid phase
4
Q
hydrophobic chromatography
- what is the solid phase
- what binds to it
- what elutes
- how to elute binded protein
A
- solid phase is hydrophobic
- high [salt] = high hydrophobic effects
- start with high [salt] buffer
- more hydrophobic proteins bind
- less hydrophobic proteins elute
- then decrease [salt] to elute more hydrophobic proteins
5
Q
affinity chromatography
- what is the solid phase
- what binds to it
- what elutes
- how to elute binded protein
A
- solid phase has specific ligand
- only proteins that bind the ligand interact with the solid phase
- elute by flowing in free ligand in mobile phase - competes for binding with protein causing a shift in equilibrium, causing protein to elute in mobile phase
6
Q
size exclusion chromatography
A
-small proteins elute last
-large proteins elute first
-cut-off = maximum size (MW) that will enter any pores-
everything larger elutes first and elutes together in the void volume.
7
Q
what is the cut off size for a particular column
A
volume of resin + the type of resin
8
Q
Ve
A
elution volume
-each protein has its own Ve
9
Q
Vo
A
void volume
10
Q
what is the equation for estimating the MW using size chromatography?
& can depend on what
A
Ve/Vo
- unique for each protein on a particular resin
- also depends on shape (sphere vs rod - don’t appear to have same MW but can - based on how elute from colum)
