Proteins 3 Flashcards
types of chromatography
size exclusion
hydrophobic interaction
ion exchange
affinity
reverse phase
how does size exclusion chromatography work
Pores in beads exclude larger molecules but smaller ones are
retarded - i.e. the volume in the column is larger for smaller
molecules
why monitor at 280nm?
thats where aromatic amino acids such as tryptophan and tyrosine absorb
Things that can degrade resolution (i.e. broaden peaks);
high solute concentrations, high flow rate, large sample volume, column needs to be repacked.
size exclusion chromatography issues
charge and shape issues
main detergents used
OG - octyl glucoside
DDM
Cymal - 5
Triton X 100
main thing to think about when using detergents
CMC
size exclusion pros
1) Can use high salt
2) Can use detergent
(below CMC)
3) Sizing often works to
some degree
4) Probably the only way
to separate oligomers
of the same protein (ehhh)
size exclusion cons
1) Dilutes the sample
2) Relatively slow
3) Slow turnaround
4) Resolution-variable
5) Sensitive to [Protein]
6) Low loading volume
ion exchange chromatography
a charged column grabs other charged proteins
two positively charged column types
Mono Q (FPLC, high resolution, comes as pre-packed columns)
DEAE, diethylaminoethyl cellulose
two negatively charged columns
Mono S (FPLC, high resolution, comes as pre-packed columns)
CMC, carboxymethyl cellulose
two kinds of buffers for ion exchange chromatography
positive and negative
when making phosphate buffer, do you need to mix all forms of phosphate to get to a certain pH?
NOOOOO
ion exchange pros
1) Can concentrate sample
2) Can use with detergents
(non-ionic)
3) Can resolve very well
4) Relatively fast (e.g. <1 hour
per run start to finish with
FPLC)
ion exchange cons
1) Does not work with
proteins that require
salt for solubility.
hydrophobic interactions are strengthened in
high salt
in hydrophobic interaction, elution occurs as
salt is decreased
problem with hydrophobic interaction
protein may never come off
natural affinity chromatography means
taking proteins from the natural source and purifying with small molecular weight ligands such as GTP, ATP, NAD(P), etc.
pros of natural affinity chromatography
1) Can concentrate the sample
2) Can have high yield - depending
upon contaminants
3) Can be a crude way to look at
protein binding - e.g. does a
mutant block binding.
cons of natural affinity chromatography
1) Can be a common ligand that
binds lots of stuff.
2) Linker to beads is critical.
Some linkages will prevent
binding to protein.
3) Elution can be costly. E.g.
maybe ATP is the only way to
elute ($$$$)
4) Usually not one step
purification.
examples of expression affinity tags
his tag
maltose binding protein
glutathione S transferase
antigens
how to remove a tag
can use Tobacco etch virus protease (TEV) to cut at a specific site
