lecture 3 Flashcards
Chromatography can be applied to which elements within chasm
1- charge
2- hydrophobicity
3- Affinity
4- Molecular weight
Ion exchange theory works on the basis of?
Overall charge can be determined if we know the sequence
Surface charge vs overall protein charge?
may be different
What type of interaction is occurring in ion exchange chromatography?
electrostatic
Matrix within Ion exchange chromatography contains
ionised groups
Anion exchange therefore?
exchanges anions
matrix- pos
proteins-neg
Cations charge
cats pur positive
Strength of the interaction between the ionised groups and the protein is due to?
number of ionic charges the proteins have
separation due to charge
Matrix functional group examples
Anion exchanger - Diethylamino ethyl (DEAE) - Quaternary ammonium Cation exchanger - Carboxymethyl - Methyl sulphonate
Stronger anions and cations
will interact over a larger range
issue with really strong or really weak binders?
will bind too much or not enough
Issue with anion and cation exchanges
may not be the appropriate pH
Conditions for adsorption?
Low ionic strength buffer
pH to generate opposite charge to ion exchange resin (know pI)
pH
protonated (positively charged)
pI for a cation exchanger
- high (neg can bind to positive)
How to choose buffer pH
one pH unit away from the pI
Choose a suitable buffer pH for a cation exchanger when the pI=8.5
Can choose 1 pH unit above or bellow
- 9.5- too high (alkali)
- 7.5 better due to this being a cation exchanger (therefore your ions will be pos charged)
Elution method ?
change in ionic strength
Highly charged ions will come off?- elution
last
What type of gradient is drown in elution
stepwise
gradient
What needs to be maintained during elution
pH as this may change the proteins ability to bind
Choose gradient such that?
they ate separated
Elution by changing the pH
Molecules with the same isoelectric point are focused in narrow bands during the separation.
What type of gradient in the collum- Elution by changing the pH. Usually is ?
pH gradient
usually low to high
Focussing increases?
resolution
when might we want to use the focussing technique to separates proteins?- range ?
isoforms
eg haemoglobin
usually over a range of 1 and a Half pH unit
Advantages of ion exchange chromatography?
1- Load large volumes, elute small (concentrating)
2- High capacity, good resolution
Disadvantages of ion exchange chromatography?
denaturation at extreme pH
product in high salt
pH gradient technically difficult to produce
Seperation due to?- Hydrophobic interaction chromatography
differing surface hydrophobicity
Hydrophobic interaction chromatography promoted by?
Salting out
In low salt?
water cages form around hydrophobic patches
The most hydrophobic will elute?
last
In high salt?- hydrophobicity
Water cages removed, bind to ions
how might we disrupt the interaction between the protein and the column- elution?
- reducing salt
- changing salt type
- adding detergent
- changing pH
- reduce temperature
Affinity chromatography relies on?
Isolate using specific property of protein
e.g. binding site
Affinity compound attaches to ?
elution and purification from this method?
inert matrix
binds only the seared protein
one step elution
3 fold purification
by what bonds are ligands attached to the matrix?
covalently
Ligands in affinity chromatography are _____specific
bio
Examples of molecules raised against proteins - affinity
- substrate enzyme
- inhibitor
- cofactor
- antibodies
Group specific examples?
Protein A or G (antibody)
Lectins (glycoproteins)
Pseudo specific
Dyes mimic cofactors
affinity equation?
Ka= [PL]/[P][L]
P+ L»> PL
Dissociation?
Kd= [P][L]/[PL]
Kd value in which is strong enough to adsorb
” to elute
Kd<10^-4
Kd>10^-8
Staphylococcal nuclease example
Binds to analogue
Eluted by changing the pH, therefore no longer charged and is washed off
How does protein stability need to be considered in affinity chromatography?
‘Spacer arm’ needed to increase the distance from the interaction area.
To prevent delocalisation during purification (preserves affinity)
Methods of elution- Affinity
- large concentrations free ligands (not bound to the matrix) - cost
- pH
- Salt (detergent)
Affinity tags usually bind to ??
Metal ions within the matrix
Affinity tags are tagged in what sense ??
tagged recombinantly (using a vector to produce)
examples of affinity tags
maltose-binding protein
- or (His)n (where n≥6)
within the matrix what is present?- affinity tags, EXAMPLES
Metal associates, part of coordination sphere
Ni2+- example
(his)n tag binds with?
imidazole
strongly interacts with the ni which causes the protein of interest with the his tag to dissociate
Advantages vs disadvantages of affinity chromatography
> 95% purity in one step
- Tagged recombinant, proteins promote solubility
disadvantages:
- finding attachable specific ligand
By adding larger tags
help correct protein folding
gel filtration assumes?
all proteins have the same shape and therefore are separated only due to their size
in the case of gel filtration what type of matrix ?
solid matrix
pourous matrix
conditions in gel filtration?
same conditions
characteristics of gel filtration?
Non-adsorptive, gentle
Proportion of pore occupied by a protein?
Kav = (Ve- Vo)
(Vt - Vo)
Ve- excluded vol of protein
Vo= void volume
Vt- total volumeKav us proportional to?
log (mw)
proteins come off in??- GF
voids
small come off last
beads must be able to ____ ___ _______ therefore ?
encompass the protein, therefore there are a range of bead sizes and materials
Peak width is due to ?
size of beads
speed of flow
advantages and disadvantages of gel filtration?
- simple
- gentle (conditions as required by the protein)
- desalting
dis - long columns - Time consuming - small samples diluting
Gel filtration is often referred to as a?
polishing step
The matrix is usable composed of
commonly agarose, cellulose or dextran,
is milled into beads of various sizes and porosity,
with different optimal separation ranges.
Maximise the final yield?
Minimise the step number
Low capacity vs high capacity methods?
low cap:
Gel filtration
High cap:
Ion exchange
Affinity chromatography
salting out (ammonium sulphate)
High resolution methods vs low resolution methods
high res:
Ion exchange
Affinity chromatography
Gel filtration
low resolution: salting out (ammonium sulphate)
detection of impurities
- SDS-PAGE
- IEF/Western blot/MS
Deacetoxycephalosporin c synthase method of protein purification?
- Lysed by sonication
2. centrifuge to clarify
3. Ion exchange
4. hydrophobic interaction (add low salt
5. Gel filtration
MBP-tagged protein 3 steps to purifications
- Maltose- bound column
- Gel filtration
- SDS-PAGE analysis
Fab fragment example
- cell lysed with sucrose
- DNAse added
- Cation exchange
- salting out
- IE with shallow gradient
recombinant myoglobin ?
express in ecoli
soluble fraction removed
affinity chromotograohy
size exclusion chromatography
Activity assays are ______ to your protein
pros
cons
Tailored
pros: rapid, reproducible, specific, cheap, sensitive
cons: colourmetrix, destructive and reduces yield
Specific assay examples? general names given
- activity assay
- binding assay
- detection of impurities
Monitor purification two method names?
- adsorption at 280nm
- Bradford assay
Concentration of protein needed for absorption at 280nm method?
- 0.1 mg/ml
absorption at 280nm method
PROS AND CONS
PROS: rapid and non destructive non quantitve (e unknown) and nucleic acids interfere
Bradford assay range of volumes
1-2 ul
pros and cons of the Bradford assay?
pros: simple, rapid. fewer interferon sustances
cons: Variation In response, requires a calibration curve
Specific activity equation?
= Enzyme activity/ unit mass protein
yield=
Total activity after nth step x 100/ Total enzyme activity in initial sample
purification factor equation?
Specific activity after the nth step/ Specific activity of initial sample
recombinant myoglobin analogy names
ELIO LIKES CUMING AND SCREMING
Ecoli expression
- LYSE
- CENTRIFUGE
- affinity
- Size exclusion
Deacetoxycephalosporin c synthase analogue
STOP CUMMING IN HER GAF
- Sonnication (ecoli)
- Centrifuge
- ion exchangge
- hydrophobicicty chrom
- Gel filtration
MBP -tagged protein analogue
Maxine Gee sexy
- Maltose binding column
- Gel filt
- SDS (analysing)
FAB Fragment analogue ?
llamas drive cars singing immensely
- Lysed (sucrose)
- Dnase
- Cation exchanger
- Salting (wash with low salt)
- IE (shallow gradient)
Bradford assay?
The Bradford protein assay is used to measure the concentration of total protein in a sample
