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ProteinPurification
ProteinPurification
CarolineRitchie(carolinedotritchie103atgmaildotcom)
IowaStateUniversity,UnitedStates
DOI http://dx.doi.org/10.13070/mm.en.2.134
Date lastmodified:20150902originalversion:20121117
Citeas MATERMETHODS20122:134
Abstract
Anindepthreviewofcolumnchromatographyforproteinpurificationandsurveyresultfrom305
formalpublications.
Purified proteins are required for many experimental applications, including structural studies and in
vitro biochemical assays. Proteins can be obtained from tissue or, more often, by their overexpression in a
modelorganism,suchasbacteria,yeast,ormammaliancellsinculture.Proteinpurificationinvolvesisolating
proteins from the source, based on differences in their physical properties. The objective of a protein
purification scheme is to retain the largest amount of functional protein with fewest contaminants. The
purificationschemeofaproteinmustbeoptimizedtocompletethisprocessintheleastnumberofsteps.
The
article
reviews
four
types
of
column
Figure1.ATrisbufferedsolutioncontainsTris
baseanditsconjugateacid.ThepKaofTrisat
25Cis8.06,indicatingthatatpH=8.06,50%of
theTrisisprotonated(initsacidicform)and50%
isdeprotonated(initsbasicform).
DevelopingaProteinPurificationScheme
The most important consideration in the development of a protein purification scheme is the downstream
applicationofthepurifiedprotein.Boththequantityandpurityoftheproteinmustbesufficientforexperimental
analysis.Additionally,informationaboutthebehavioroftheproteinmustbetakenintoconsideration,aswell
foldedandfunctionalproteinisrequiredfordownstreamstudies.Duringpurificationandsubsequentstorage,
manyprocessescanoccurthataffectproteinquality:proteinunfolding,aggregation,degradation,andlossof
function.Carefulplanningtopurifyproteinasquicklyaspossibleandunderthemoststabilizingconditionswill
maximizethechanceofasuccessfulpurificationscheme.
BufferingComponent
Thesolutionconditionsofaproteinateachstepofthepurificationschemeareessentialinmaintainingprotein
stabilityandfunction.ProteinsshouldbekeptinawellbufferedenvironmenttopreventsuddenchangesinpH
thatcouldirreversiblyaffecttheirfolding,solubility,andfunction.
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Abufferisasolutioncontainingaconjugateacid/basepair.ThepHrangeofabufferis
basedonitspKa,definedasthepHatwhich50%ofthemoleculesareintheiracidic
formand50%areintheirbasicform(Figure1).Ageneralruleregardingbuffersisthat
thepHofthebuffersolutionshouldbewithin1.0pHunitofthepKainordertoprovide
appropriate buffering capacity. This ensures that there is a sufficient amount of the
molecule in both its acidic and basic forms to neutralize the solution in case of H+ or
Figure2.An
Aktaprimeplus
systemforthe
automated
chromatographic
separationof
proteins.From
GE.
OH influx. Thus, buffers prevent pH changes that could negatively affect protein
stability.
A good buffer must exhibit the
followingcharacteristics[1]:
1.Watersolubility
2.Chemicalstability
3.Highbufferingcapacityat
desiredpH
4.Compatibilitywith
analyticaland
experimentalapplications
5.Compatibilitywithother
solutioncomponents
Manycomponentscanserveasbiologicalbuffers.The
most commonly used buffering components have a
nearneutralpKa,astheycanbeusedataphysiological
pH. Four of the most common biological buffers are
listedinTable1,alongwiththepHrangeatwhichthey
Figure3.NiNTAligandcovalentlyattachedtoa
crosslinkedagarosematrixfortheselective
purificationofpolyhistidinetaggedproteins.
HistidineresiduescancoordinatetotheNi2+ion
byreplacingtheboundwatermolecules
(indicatedbyredarrows).Imidazoleorfree
histidinecanthenbeusedtoelutetheprotein,
throughtheirabilitytocoordinatewiththeNi2+ion
anddisplacetheboundprotein.FromBioKe.
pHrange
Advantagesand
Disadvantages
pHisnot
dependenton
temperature
Inexpensive
Phosphate
5.88.0
Transparentin
UVrange
Cannotbeused
withdivalent
cations
https://www.labome.com/method/ProteinPurification.html
Inadditiontoanappropriatebufferingsystem,solutions
used during protein purification from lysis to storage
often contain many other components that play a role
infacilitatingproteinpurity,stability,andfunction.
Protease inhibitors are often added to the lysis buffer
andinearlystepsofthepurificationschemetoprevent
degradation of the target protein by endogenous
proteases. These are generally not needed toward
later stages of the purification, as most or all of the
contaminatingproteaseshavebeenseparatedfromthe
protein of interest. Metal chelating reagents, such as
EDTA or EGTA, are often added to the storage buffer.
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Cannotbe
autoclaved
pHissomewhat
MOPS
6.57.9
dependenton
temperature
Highbuffering
ThesemetalchelatorsbindtoMg2+and,thus,prevent
cleavage of the purified protein by contaminating
metalloproteases. Other additives are often used to
protect proteins against damage and enhance their
solubility.
capacityat
physiologicalpH
Cannotbe
autoclaved
HEPES
6.88.2
Otherfactorstoconsider
pHissomewhat
dependenton
Otherfactorsalsocontributetoproteinstabilityduringa
temperature
Canformradicals
undercertain
conditions[2]
highestyieldoffunctionalprotein.Additionally,itisoften
purificationscheme.Theleastmanipulationofaprotein
7.59.0
Caninterferewith
activityofsome
enzymes[3],[4]
Transparentin
UVrange
Table1.Mostcommonlyusedbiologicalbuffers
forproteinpurification.Buffersmaintaintheir
bufferingcapacitywithinaspecificpHrange,and
characteristicsofsomebufferingcomponents
couldinterferewithparticularchromatographic
proceduresoranalysis.Summarizedfrom
Promega,SigmaAldrich,Applichem,Embl.de.
Theprincipleofcolumnchromatographyistoseparate
alargepoolofproteinsintomanysmallerpools,some
of which are enriched in the protein of interest. While
expensive and specialized equipment is available for
column chromatography, only basic equipment is
required.
Basicequipmentforcolumnchromatography:
1.Stationaryphase:aninert
matrix,oftenwithan
attachedfunctionalgroup
Type
Function
tofacilitateprotein
interaction,usedto
separateproteins.The
choiceofstationary
Protectagainstoxidative
ReducingAgents
phaseandfunctional
damage
groupdependsonboth
thetypeof
chromatographythatis
https://www.labome.com/method/ProteinPurification.html
CommonlyUsedReagents
2mercaptoethanol
(BME)
Dithiothreiotol(DTT)
Tris(2carboxyethyl)
phosphine(TCEP)
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beingperformedandthe
methodbywhichitwillbe
carriedout.
2.Column:acylindrical,
Inhibitendogenous
glassreservoiravailable
ProteaseInhibitors
proteasesfrom
invariouslengthsand
degradingproteins
diameters.Columnscan
bepurchasedprepacked
withastationaryphase
andreadytoattachto
automated
chromatographysystems
Inactivate
MetalChelators
(discussedinPart2of
metalloproteases
thissection)orcanbe
boughtemptyformanual
packing.Differenttypesof
columnsarerequiredfor
automatedversusgravity
Stabilizeproteinstructure
Osmolytesflowchromatography.
andenhancesolubility
Leupeptin(serineand
cysteineprotease
inhibitor)
PepstatinA(asparticacid
proteaseinhibitor)
PMSF(serineprotease
inhibitor)
EDTA
EGTA
Glycerol
Detergents(e.g.,CHAPS,
NP40,TritonX100)
3.Solvents:buffers
Sugars(e.g.,glucose,
containingadditivesused
sucrose)
forequilibrating,washing,
andelutingproteinsfrom
Salts(e.g.,NaCl,KCl,(NH4 )2 SO4
IonicStabilizers
Enhancesolubility
thestationaryphase.
Differenttypesof
chromatographyrequire
Table2.Additivescommonlyusedinproteinpurificationbuffersto
differentsolvent
increasestabilityofproteins.SummarizedfromThermoFisherPierceand
Embl.de. conditions.
4.Collectiontubes:vessels
forcollectingelution
samples.Specialtubes
arerequiredfor
automatedfraction
collectorshowever,any
tubeorvesselis
appropriateformanual
fractioncollection.
Figure4.Schematicofaffinity
purificationusingProteinA,G,or
L.A.Antibodiescontainseveral
regionsofinterest:Fc(fragment,
constant)thatisthesameforall
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5.Assaytomeasurepurity:
anapproachfor
determiningtherelative
amountofaspecific
proteintothetotalprotein
inasample.Thefractions
containingtheproteinof
interestmustbe
determinedaftereach
stepbeforeproceedingto
thenextstepinthe
purificationscheme.
Somecommonmethods
ofanalyzingpuritywillbe
discussedinalater
section.
SystemsforColumnChromatography
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proteinsofthesameclass,Fv
(fragment,variable)thatoffers
specificityforeachantibody,and
Fab(fragment,antigenbinding)
thatactuallycontactsthespecific
antigen.B.Specificclassesof
antibodiescanbenonselectively
purifiedthroughaffinitypurification
inwhichtheligand(ProteinA,G,
orL)isconjugatedtoamatrix.C.
ProteinA,G,orLcanalsobe
usedtopurifyspecificproteins.In
thiscasetheantibodywillserve
asanintermediateligand
providingselectivityforitsantigen.
TypeofSystem
Advantages
Disadvantages
Canletrunbyitself
Oftencoupledtoan
absorbancedetector
Automated
Canprogram
equilibrationand
washsteps
Easytosetupa
gradientforelution
Requiresspecific,
costlyequipment
Maximumflowrate
isdependenton
pressurelimitof
column
Veryreproducible
Lessexpensive
Theuserhasmore
control
GravityFlow
Canmake
adjustmentsduring
run
appropriate chromatographic
method(s) and the order of
Morelaborintensive
Flowroteislimited
bygravity
Table3.Systemsforthechromatographicseparationofproteins.
SummarizedfromGE.
binding of a protein to a
matrixbound ligand. The
Typeof
Chromatography
robust
Affinity
purification
https://www.labome.com/method/ProteinPurification.html
Separates
ProteinsBy
Aspecific
interaction
BindWith
Nocompeting
ligand
EluteWith
Competingligand
(specific)
conditionsthat
disrupt
protein/protein
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ProteinPurification
interactions(non
specific)
on
the
application,
IonExchange
Netsurfacecharge
Lowionicstrength
Highionicstrength
Increased(cation
exchange)or
decreased(anion
exchange)pH
Hydrophobic
Interaction
Hydrophobicity
Highionicstrength
Lowionicstrength
SizeExclusion
Hydrodynamic
radius
Table4.Thefourmostcommontypesofcolumnchromatographyusedin
proteinpurification.
Figure5.Thenetchargeonaproteinis
influencedbythepHofitssolvent.AtpH=pI,the
proteinhaszeronetchargeand,therefore,will
notbindtoacationexchangeorananion
exchangestationaryphase.AdjustingthepH
aboveorbelowthepIoftheproteinwillleadtoa
netcharge,andproteinbindingtoeitherananion
exchange(pH>pI)oracationexchange(pH<
pI)stationaryphase.
proteins and the stationary phase. The bound protein is then eluted with a buffer containing a competing
molecule or conditions that disrupt all protein/protein interactions. Competing molecules bind to the ligand,
displacing the protein of interest. This competing molecule is typically removed from the protein of interest
eitherthroughanotherchromatographicprocedureordialysis.Methodsforelutingproteinsfromthestationary
phase by disrupting all protein/protein interactions include adjusting the pH or ionic strength of the buffer.
These methods can affect protein stability, and it is suggested that the eluted protein be immediately
neutralized or diluted to minimize protein damage. For some forms of affinity chromatography, alternative
elutionconditionshavebeendescribedtomaximizetheyieldoffunctionalprotein[15,16].
Proteinto
Purify
Antibody
(antigen
specific)
Polyhistidine
Ligand
Antigenic
peptide
EluteWith
Indesigningaplasmidforproteinexpression,affinity
tags can be inserted on either the N or Cterminus
Freepeptide
Imidazoleor
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commontagsandtagrelateddiscussions.
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ProteinPurification
taggedprotein
Ni2+orCo2+
freehistidine
FLAGtagged
protein
FLAG
specific
antibody
FLAG
peptideor
lowpH
GSTtagged
protein
Reduced
glutathione
Free
glutathione
Myctagged
protein
Mycspecific
antibody
LowpH
Antibody
(classspecific)
ProteinA,
G,orL
Extremesin
pH
DNAbinding
protein
Heparin
Highionic
strength
Table5.Examplesofselectiveandnonselective
formsofaffinitychromatographywiththefunctional
group(ligand)usedforspecificityandtypical
elutionconditions.SummarizedfromThermo
FisherPierceandGE.Seebelowforthecommon
suppliersfordifferenttypesofcolumnsbasedona
Labomesurveyofover200publications.
beaffinity
purifiedin
a
non
selective
manner.
In non
selective
Figure6.Ionexchange
chromatography.Proteinsarebound
toachargedstationaryphaseatlow
ionicstrength.Theboundproteins
canbeelutedeitherbyincreasing
theionicstrengthofthebufferorby
adjustingthepH.
purification, the ligand attached to the stationary phase binds to a group of proteins with similar binding
partners.AnexampleofnonselectiveaffinitypurificationisinthepurificationofDNAbindingproteins.Heparin
mimics DNA both in its structure and its charge, and can be used as the ligand for the affinity purification of
DNAbinding proteins. While all DNAbinding proteins could theoretically bind to this stationary phase, most
other proteins will flow through without binding, leading to sufficient enrichment of the protein of interest.
Another example is the enrichment of antibodies by the binding of their constant (Fc) region to the ligand,
ProteinA,G,orL.GEHealthcareproteinAaffinitycolumnsareacommonchoice[1719],asisproteinG[20].
Usingasimilarmodeofbinding(anantibodyboundtoaProteinA,G,orLligand),theantigenbinding(Fab)
regionoftheantibodyisstillavailableforbindingtoitsspecificantigen.Thus,specificproteinscanbepurified
basedonthespecificinteractionbetweenthecoupledligand/antibodyandtheantibody/antigen.
CommonproblemsandtroubleshootingarelistedinTable6.
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IonExchangeChromatography
(IEX)
Problem
Proteindoesnotbind
proteins
and
Proteindoesnotelute
and
(2)
cation
Lowresolution
is commonly used as an
intermediate step in a protein
purification scheme however,
it can yield high resolution for
some proteins when used
earlier or later during the
purification.
All proteins exhibit a net
Solution
Checkplasmidsequence
Tagwasnottranslatedoris
ormovetagtoadifferent
notaccessible
location
Ionexchangechromatography
(IEX) separates proteins
based on their net surface
Cause
Bindingconditionsarenot
appropriate
Adjustbufferconditions
Notenoughtimewas
allowedforbinding
Decreaseflowrateorstop
columntoallowincubation
Affinitybetweenligandand
tagisveryhigh
Increaseconcentrationof
competitor(specific)or
stringencyofconditions
(nonspecific)
Proteinaggregatedon
column
Adjustbufferconditionsfor
moreproteinstability
Flowrateiseithertoofast
ortooslow
Adjustflowrate
Columnwasnotwashed
sufficiently
Washwithhigher
stringencybufferClean
stationaryphaseaccording
tomanufacturer
Proteinaggregatedon
column
Adjustbufferconditionsfor
moreproteinstability
Elutionconditionsarenot
stringentenough
Increaseconcentrationof
competitor(specific)or
adjustconditions(non
specific)
Proteinisunfoldedor
aggregated
Adjustbufferconditionsfor
moreproteinstability
Proteinlosesactivityduring
Acofactorrequiredfor
procedure
activitywasremoved
duringpurification
Addcofactor
Table6.Troubleshootingaffinitychromatography.SummarizedfromGE.
attachedmodifications.ThenetchargeofaproteinisinfluencedbythepHofthesolventthatitisdissolvedin,
assolventsexchangehydrogenionswithproteins.Theisoelectricpoint(pI)ofaproteinisthepHatwhichthe
proteinhasnonetcharge.AtapHabovethepI,aproteinwillhaveanetnegativecharge,whileapHbelow
thepIwillleadtoanetpositivecharge.Thus,thepHofthesolventcanbeadjustedtofacilitatebindingtoIEX
ortopromoteelutionofaboundprotein.
Theoretically, all proteins can bind to both cation exchange and anion exchange if the pH of the buffer is
adjusted accordingly. However, for protein purification, the stability of the protein is the most important
consideration in choosing purification conditions and, thus, the most appropriate column for protein binding.
Therefore,itisnecessarytodeterminewhetherconditionsrequiredforbindingtoeitherformofionexchange
chromatography affect protein stability and function. Typically, conditions for binding to one type of ion
exchangeismoresuitableforaparticularproteinthantheother.
Knowledge of a protein's isoelectric point can aid in determining the most appropriate type of ion exchange
chromatography. Online tools are available for calculating the theoretical pI of a protein
EXPASY
. These
calculationsarebasedentirelyontheaminoacidsequenceofaproteinanddonottakethethreedimensional
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Figure7.Hydrophobicinteraction
chromatography.Athighionic
strength,proteinsarepartially
desolvated,causingthemtoadopt
alternateconformationsinwhich
normallyburiedhydrophobic
residuesaremoreexposed.These
residuescanthenformhydrophobic
interactionswiththehydrophobic
functionalgroupsconjugatedtoa
matrix.Loweringtheionicstrength
causestheproteintorefoldintoits
nativeconformation,buryingits
hydrophobicresidues.This
decreaseshydrophobicinteractions
betweentheproteinandstationary
phase,facilitatingproteinelution.
forlargerproteins,porousmatricescausealossinresolutionbasedonsizeexclusioneffects[28].
The four charged functional groups most commonly used in
IEXareshownbelow.Theseareclassifiedaseitherstrongor
weakexchangers,withstrongexchangersbeingionizedovera
widerpHrangethanweakexchangers.
Anionexchange(positivelychargedstationaryphase):
1.quaternaryammonium(Q)stronganionexchanger
2.diethylaminoethyl(DEAE)weakanionexchanger
Cationexchange(negativelychargedstationaryphase):
1.methylsulfonate(S)strongcationexchanger
Figure8.Proteinelutedfroma
hydrophobicinteractioncolumnwitha
decreasingsalt(ammoniumsulfate)
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2.carboxymethyl(CM)weakcationexchanger
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gradient.Fractionswereanalyzedforboth
totalproteincontentandactivityspecific
totheproteinofinterest.Thepeak
centeredatfraction45containsthe
proteinofinterest,asindicatedbyprotein
activity.From
http://www.insectscience.org/9.04/.
Figure9.Amixtureofthreeproteinsofvarying
hydrodynamicradiusisloadedontoasize
exclusioncolumn.Largeproteinselutefirst,as
theyareunabletoentertheporesofthematrix
andhaveastraightforwardpaththroughthe
column.Smallerproteinscanenterthepores,
haveamoreconvolutedpathand,thus,take
longertotraversethematrixandelutefromthe
column.
high ionic strength buffer and,
Problem
Cause
Solution
Runmoreequilibration
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Proteindoesnotbind
ProteinPurification
Columnwasnot
equilibrated
bufferthroughcolumnand
reloadprotein
ionexchangechromatography
Ionicstrengthofbinding
bufferistoohigh
Lowerionicstrengthof
buffer
AdjustbufferpH(lowerfor
cationexchange,higherfor
anionexchange)
pHisnotfarenoughfrom
pI
Proteindoesnotelute
Lowresolution
ammonium
sulfate
Ionicstrengthofelution
bufferistoolow
Increaseionicstrength
Proteinaggregatedon
column
Adjustbufferconditionsfor
moreproteinstability
Flowrateiseithertoofast
ortooslow
Adjustflowrate
Columnwasnotwashed
sufficiently
Washwithahigherionic
strengthbufferClean
stationaryphaseaccording
tomanufacturer
Proteinaggregatedon
column
Adjustbufferconditionsfor
moreproteinstability
Proteinisunfoldedor
aggregated
Adjustbufferconditionsfor
moreproteinstability
Proteinlosesactivityduring
Acofactorrequiredfor
procedure
activitywasremoved
duringpurification
proteinsbyprecipitatingsome,
but not all, proteins with salt.
HICissometimesapplicablein
early steps of a purification
scheme or as a final step in
theremovaloftraceimpurities
fromtheproteinofinterest.
Table7.Troubleshootingionexchangechromatography.Summarized
fromGE.
hydrophobic
residues.
Proteins that bind to the
stationary phase adopt their
native conformation as buffer
with a lower ionic strength is
added. This decreases the
exposure of hydrophobic residues that can interact with the stationary phase, and facilitates elution from the
column.Forproteinsthatcanrefoldspontaneouslythroughadecreaseinionicstrength,HICcanbeavaluable
chromatographicmethodforpurification.
Typically, the ionic strength of the binding buffer should be as low as possible to bind the protein of interest,
while preventing its precipitation. If the ionic strength required for protein binding is high and causes
precipitation of the protein of interest, a lower ionic strength can be used. In this case, the chromatography
procedure can be used to separate all of the binding proteins from the protein of interest that flows through
withoutbinding.
Prior to loading the sample on the column, the stationary phase must be equilibrated with the high ionic
strengthbuffer(thesamebufferthattheproteinsamplewillbeappliedin).Thesampleisthenloadedontothe
column,thecolumniswashedextensively,andtheproteiniselutedwithalowionicstrengthbuffer(Figures7
and8).
Thestationaryphaseusedinhydrophobicinteractionchromatographyiscomposedofabasematrixofeither
crosslinked agarose or a synthetic copolymer. An alkyl or aryl ligand is then conjugated to the base matrix,
providingspecificityforhydrophobicmolecules.
Typesoffunctionalgroup:
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1.Alkylahydrocarbonchainofvariouslengthoftenabutyloroctylgroupisused.Thebindingcapacityof
thestationaryphaseisincreasedwithincreasingalkylchainlength[33].Thefunctionalgroupsbind
proteinsbasedentirelyonhydrophobicityoftheprotein
2.Arylafunctionalgroupderivedfromanaromaticringoftenaphenylgroup.Arylgroupsofferincreasing
specificity,asproteinscanalsointeractwiththefunctionalgroupthroughbasestackinginteractions.
The type and concentration of salt used for binding and elution must be determined empirically for each
protein.Additionally,therefoldingandfunctionoftheproteinmustbeensuredafteritselution.
Like other forms of column chromatography, an optimum HIC procedure can require a great deal of
troubleshooting. Adjusting buffer conditions and stationary phase are required for each protein to ensure
optimalseparation.
Sizeexclusionchromatography
(SEC)
Problem
Size
exclusion
chromatography
separates
proteins
by
their
hydrodynamic
radius,
a
property determined by both
the size and shape of the
Proteindoesnotbind
Proteindoesnotelute
[34],
unfolded
protein
molecules [35], and truncated
morereliablemethodofbuffer
exchangethandialysis,asitis
Runmoreequilibration
bufferthroughcolumnand
reloadprotein
Ionicstrengthofbinding
bufferistoolow
Increaseionicstrengthof
buffer
Proteinaggregatedatthe
ionicstrengthused
Decreaseionicstrengthof
bufferortryadifferentsalt
Ionicstrengthofelution
bufferistoohigh
Decreaseionicstrength
Proteinaggregatedon
column
Adjustbufferconditionsfor
moreproteinstability
Retentionistoohigh
Tryadifferentstationary
phasethatoffersless
retention
Flowrateiseithertoofast
ortooslow
Adjustflowrate
Columnwasnotwashed
sufficiently
Washwithalowerionic
strengthbufferClean
stationaryphaseaccording
tomanufacturer
Proteinaggregatedon
column
Adjustbufferconditionsfor
moreproteinstability
Poorretentiononcolumn
Tryadifferentstationary
phasethatoffersgreater
retention
Proteinisunfoldedor
aggregated
Adjustbufferconditionsfor
moreproteinstability
Proteindidnotreturntoits
Proteinlosesactivityduring
nativeconformation
procedure
exchanging
buffer
components. Often times,
SEC is used as a faster and
Solution
Columnwasnot
equilibrated
Lowresolution
duetoitsabilitytodifferentiate
betweendifferentspeciesofa
protein. Oligomeric species
Cause
Acofactorrequiredfor
activitywasremoved
duringpurification
Trybindingwithadifferent
saltoratalowerionic
strengthIncludeadditives
forproteinstability
Addcofactor
Table8.Troubleshootinghydrophobicinteractionchromatography.
SummarizedfromGE.
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compatiblewithmoresolvents
and requires less buffer. A single solvent is used throughout the entire SEC procedure, and commercially
availableSECstationaryphasesarecompatiblewithmostcommonlyusedbuffercomponents.
The type of stationary phase used and the length of the column both play critical roles in influencing the
resolution of proteins separated by SEC. Several types of stationary phases are commercially available, and
thebestoptiondependsonboththemolecularweightoftheproteinsrequiringseparationandtheconditions
underwhichtheseparationwillbeperformed.
TypeofStationary
Phase
Matrix
Features
Offersquickbuffer
exchangeandgroup
separation
Workswellfor
molecularweight
determination
Sephadex
Crosslinkeddextranand
epichlorohydrin
Autoclavable
Matrixcanshrinkin
certainsolvents
Specifictypesof
sephadexare
availableforusewith
organicsolvents
Separatesmolecules
overalarge
Sephacryl
Crosslinkedallyldextran
andN,Nmethylene
bisacrylamide
molecularweight
range
Autoclavable
Workswithaqueous
andorganicsolvents
Highrecovery
Separatesmolecules
Sepharose
Crosslinkedagarose
overalarge
molecularweight
range
Highrecovery
Cannotbeautoclaved
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1.Providesbufferexchange
anddesalting.
2.Enablestheseparationof
similarspecies(e.g.,
truncationsandoligomers)
thatmightnotbe
separatedwithother
purificationtechniques.
3.Iscompatiblewithmany
solvents.
4.Doesnotdependonany
specificproteinproperty
forretentionandelution.
DisadvantagesofSEC:
1.Performanceisvery
sensitivetocolumn
packing.
2.Canhavenonspecific
interactionsbetween
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Workswithaqueous
andorganicsolvents
Superose
Highlycrosslinked
agarose
Autoclavable
Hydrophobic
interactionsbetween
proteinsandmatrixis
possible
Compatiblewith
viscoussolvents
proteinandresin,which
decreasesresolution.
3.Offerslowresolutionfor
complexproteinmixtures.
4.Samplemustbeloadedat
asmallvolumefor
adequateresolution.This
canbeproblematicfor
proteinsthatprecipitateat
highconcentrations.
Optimizing SEC conditions to
Workswithaqueous
andorganicsolvents
Superdex
Crosslinkedagarose
anddextran
Autoclavable
Highresolution
Highrecovery
Table9.Typesofcommerciallyavailablestationaryphasesforsize
exclusionchromatographyandfeaturesofeach.SummarizedfromGE
andSimgaAldrich.
1.Minimizethesample
volume.Thesmallerthe
volume,thelessdiffuse
theelutedfractionswillbe.
2.Addsalttothebuffer.A
smallamountofsaltwill
helppreventnonspecific
interactionsbetweenthe
proteinandstationary
phase.Thiswillallowall
proteinmoleculestorun
consistentlyoverthe
lengthofthecolumn.
3.Useamoderateflowrate.
Runningacolumntoo
quicklywillnotallowtime
forsmallmoleculesto
enterthematrixpores,
whileaflowratethatistoo
slowallowsmoretimefor
samplediffusion.
4.Ensurethatsampleand
solventviscositiesare
similar.Adjustsample
conditionsto
approximatelymatchthat
oftherunningbuffer.
5.Adjustthecolumnlength.
Acolumnthatistooshort
willnotallowadequate
proteinseparation.
Alternatively,acolumnthat
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ProteinPurification
istoolongwillleadto
diffusionoftheprotein
sample.
6.Repackthecolumn.
Columnpackingcanhave
ahugeeffectonprotein
resolution.
Ifparticlesarenotwelldispersedorifairbubblesaretrappedinthecolumn,navigationthroughthestationary
phasewillnotoccurproperly.Additionally,ifthecolumnisallowedtorundry,itmustberepacked.Poorcolumn
packingisoftentoblameforunexplainedlowresolution.
BecauseSECdoesnotseparateproteinsbasedoninteractionswithafunctionalgroup,allproteinsareeluted
under the same conditions and, thus, resolution can only be obtained for proteins of very different
hydrodynamic radius. Therefore, SEC is not an appropriate chromatographic method for initial stages of a
purification scheme when there are many contaminating proteins. SEC does, however, offer a quick and
reliablemethodforremovingsaltsorsmallmoleculesfromasamplebetweenearlyorintermediatestagesof
thepurification.Inthefinalstagesofpurification,whenonlytracecontaminantsexist,SECisavaluablemethod
forproteinisolationandexchangeintostoragebuffer.
ProteinElutionandAnalysis
MethodsofProteinElution
Proteinsthatbindtoastationaryphaseareelutedwithsolventconditionsthatdisruptthebindinginteractions.
Theseelutionconditionsvarybothbytypeofchromatographyandpropertiesoftheproteinofinterest.There
are three general methods of protein elution: batchwise elution, stepwise elution, and linear gradient elution.
The best method to choose depends on the type of chromatography being performed and the required
resolution.
1.Batchwiseasingleelutionconditiondisplacesallboundproteinsinasinglestep.Thismechanismworks
bestforchromatographicproceduresbasedonaveryspecificinteraction(i.e.,affinitychromatography).
Batchwiseelutiondoesnotofferanyresolution,butitisidealforgettingridofcontaminantsveryquickly.It
requirespriorknowledgeofbufferconditionsrequiredfordisplacementoftheproteinofinterest.
2.Stepwisemultiplebatchwiseelutionsareperformedsequentially,withmorestringentconditionsineach
step.Instepwiseelution,thenumberoffractionscollectedisdependentuponthenumberofsequential
elutionconditions.Stepwiseelutionprovidesbetterresolutionthanbatchwiseelution,butpoorerresolution
thanlineargradientelution.
3.LinearGradientmultipleconsecutivefractionsarecollectedwhileelutionconditionsareadjustedina
linearfashion.Alineargradientoffersthehighestresolutionforionexchangechromatographyand
hydrophobicinteractionchromatography.Typically,alargenumberofconsecutivefractionsarecollected.
Size exclusion chromatography does not require any of these elution methods, as no interactions occur
between the protein and the stationary phase. Protein is loaded onto the column, and a large number of
fractionsarecollecteduntilallproteinsareeluted,withoutalteringbufferconditionsthroughouttheprocedure.
Ideally,theelutionbufferofacolumniscompatiblewiththesubsequentcolumn,eliminatingtheneedforbuffer
exchangeordialysisbetweenpurificationsteps.
AnalysisofFractionPurity
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Aftereachchromatographicseparation,fractionsmustbeanalyzedtodeterminethefractionsthatcontainthe
proteinofinterestandtherelativepurityofeachofthosefractions.Thisanalysisisnecessaryaftereachstep
todecidewhichfractionsshouldbepooledforsubsequentuse.Todeterminepurity,anassayisrequiredthat
canmeasuretheamountofaspecificproteinrelativetotheamountoftotalprotein.Thefollowingassaysare
routinelyusedforpurityanalysis:
1. Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis(SDSPAGE)adenaturinggelthatseparates
proteins based on size. Commercially available stains allow for a visual representation of all proteins in
the sample, offering a qualitative assessment of protein purity (Figure 10). SDSPAGE is not ideal for
highthroughputanalysisoffractionsandcantakeseveralhourshowever,itismostoftenusedbecause
itiseasy,inexpensive,andsuitableforanyprotein.
2.Spectroscopyamethodforanalyzingopticalpropertiesof
proteins.Thistechniqueforanalyzingproteinpurityisonly
suitedforproteins,suchascytochromeP450s,thathavea
uniquespectroscopicfeature.Proteinsinthisfamilyabsorb
lightatawavelengthwhereotherproteinsdonot(around420
nanometers[37]),soacomparisonofabsorbanceat420
nanometersversus280nanometers(thewavelengthatwhich
allproteinsabsorb),canprovideaquantitativemeasureof
proteinpurity.Thismethodisfastandhighthroughput,butonly
suitableforsomeproteins.
3.ProteinActivityanenzymatictestthatdependsontheprotein
ofinterest.Thismethodofassessingproteinpurityisoften
coupledwithanotherformofproteinconcentration
determinationtocalculateactivityrelativetototalprotein
concentration.Activityassaysareonlysuitableforproteins
withactivitythancaneasilybemonitoredinahighthroughput
Figure10.SDSPAGEofsamples
format,suchasproteases.Forsomeproteins,activityassays
collectingduringaproteinpurification provideafastandreliablemethodforproteindetection.Activity
scheme.Gelisstainedforthe
measurementisoftenidealforenzymes,becauseproteinthat
visualizationofallproteins.From
haslostactivitycanbeexcludedfromsubsequentuse.
http://www.omicsonline.org/.
PurifiedProteinStorage
Whenproteinsaredeemedpureenoughforuseinexperimentalstudies,theyshouldbestoredappropriately.
Theselectionofafinalstoragebufferisjustasimportantastheselectionofbuffersusedduringthepurification
scheme,andshoulddependonstabilityoftheproteinandconditionsrequiredforthedownstreamapplication
ofthepurifiedprotein.Oftentimes,sizeexclusionchromatographyisselectedasafinalstepinthepurification
scheme,asthestoragebuffercanbeusedinthischromatographicsteptoeffectivelyexchangethebuffer.The
purefractionscanbepooledforimmediatestorage.Alternatively,thefinalpooledfractionscanbedialyzedinto
theselectedbufferbeforestorage.
Proteinstorageconditionsdependontheproteinofinterest,andshouldbeoptimizedsotheproteinmaintains
structuralandfunctionalstabilityoverlongperiodsofstorage.Additivesareoftenincludedinthestoragebuffer
to enhance the lifetime of purified proteins under storage conditions, and trial and error is often required to
determineoptimumconditions,aseveryproteinbehavesdifferently.
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