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
how is TEV normally used
1) Express protein of interest.
2) Purify with the tag (e.g. His tag and nickel affinity columns)
3) Add rTEV (usually overnight at 4°C)
4) Pass over nickel column to get rid of the rTEV protease.
affinity tags pros
1) Can concentrate the
sample
2) Can have high yield
3) Can yield high purity
affinity tags cons
1) Not all proteins can
accommodate the tags
2) Go for cheap elution
tags…
equilibrium ultracentrifugation separates via
density
non-equilibrium untracentrifugation separates via
density and size
examples of equilibrium ultracentrifugation medium
CsCl, K tartrate
examples of non-equilibrium ultracentrifugation medium
sucrose, glycerol, Ficoll-Paque
equilibrium ultracentrifugation pros
1) Can concentrate sample
2) Can yield high purity
equilibrium ultracentrifugation cons
1) CsCl can be harsh
2) Takes >24hrs
non-equilibrium ultracentrifugation pros
1) Gentler to sample
2) Can remove a great deal of
debris
3) Faster (1-2 hours)
non-equilibrium ultracentrifugation cons
1) Will not concentrate
sample as much.
2) Not as sensitive as
density purification.
the best way to look ar oligomeric state is with
analytical centrifugation
what is agarose
highly purified polysaccharide derived from agar, long sugar polymers held together by hydrogen and hydrophobic bonds
what is acrylamide
polyacrylamide gel structure held together by covalent cross-links
why should you not trust MW on SDS gels
Some proteins are VERY tightly packed and hard to reduce
even if boiled with SDS and BME
Highly charged proteins are hard to ‘neutralize’ with SDS.
May still run too fast or too slow.
Many teach to plot Rf vs Log(MW). Meh. Everything is a
straight line in a Log plot.
2D electrophoresis
First dimension, you run an IEF. Separate native proteins via charge. You
then lay that IEF strip onto an SDS-PAGE to separate denatured proteins
via size. The spots can be isolated, digested, and identified via MS
western blot chromogenic reactions
are very useful
since they do not require any special equipment for development. However,
they are not very sensitive and can be more difficult to quantify.
Alkaline phosphatase: NBT, BCIP,
Fast Red AS-MX.
Horseradish peroxidase: 4-CN, DAB, and TMB. I tend to favor these since HRP is more stable and these substrates are relatively inexpensive.
western blot chemiluminescence reactions
Reaction can be more sensitive and more quantitative. Not as easy or as fast as chromogenic and requires x-ray film, image
plate, or CCD technology.
three types of chemical precipitation
acetone
ammonium sulfate
polyethylene glycol
why not use acetone for chemical precipitation
very harsh, lots of waste
problems with polyethylene glycol
Polyethylene glycol;
Hard to get rid of - best used on viruses
ammonium sulfate chemical precipitation
Can either stabilize or destabilize protein
Can effectively concentrate from large volumes
Can use sequential cuts to remove contaminants
Easy to remove via dialysis
However, may lose protein if too dilute.
centrifigal concentrators
Wide range of volumes and MW
cutoffs
Not effective with very large
volumes
tangential flow
Like centrifuge
concentrators - pressure
pushes small MW
compounds through.
Here, the tangental flow of
material keeps the filter
from clogging.
Expensive but the only
way to concentrate large
volumes
what buffer could you use to refold inclusion bodies
glutathione redox buffer
refolding example steps
expression
isolation and denaturation of inclusion bodies
protein refolding via redox buffer
protein purification
his tag removal by TEV
size exclusion chromatography
how to detect proteins without enzymatic activity
western blots (if antibody is available)
fluorescent protein
what is the first purification method you would try with a very basic protein
E. coli expression
what is the pI of a protein and how do you measure it
pI of the protein is the pH at which the net charge of the protein is zero. it is measured via isoelectric focusing (IEF)
why would a protein band shift in apparent molecular weight in SDS page with or without a reducing agent
dimers
intermolecular or intramolecular disulfides
how would phosphorylation affect SDS page
phosphorylation would add an extra negative charge, would change movement
how would glycosylation have an effect on SDS page
smeared band because of natural heterogeneity in the sugars.
