26/09/2016

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

chromatography that are commonly used in protein

purification, and discusses the advantages,
disadvantages, and potential problems of each. This
article also reports a Labome survey on 98
publications. The survey indicates that affinity column
chromatography, mainly that based on HIS, GST,
andFLAGtags,andsizeexclusionchromatographyare
the main methods cited in the publications. GE
Healthcare is the major supplier of reagents and
instrumentsusedinproteinpurfication.

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.
https://www.labome.com/method/ProteinPurification.html

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ProteinPurification

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.

can be used, and advantages and disadvantages that

mightaffecttheirusageinproteinpurification.Typically,thesebuffersareusedatconcentrationsabove25mM
toensureadequatebufferingcapacity.
SolutionAdditives
Buffer

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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ProteinPurification

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

Additives should only be used if necessary. Trial and

error is often required to determine the specific

physiologicalpH

additives that are beneficial to a particular protein

purificationscheme.

Cannotbe
autoclaved

HEPES

6.88.2

Otherfactorstoconsider

pHissomewhat
dependenton

Otherfactorsalsocontributetoproteinstabilityduringa

temperature
Canformradicals

during its purification is always the best. Designing a

purification scheme that uses the minimum number of

undercertain

steps in the shortest amount of time ensures the

conditions[2]

highestyieldoffunctionalprotein.Additionally,itisoften

purificationscheme.Theleastmanipulationofaprotein

best to keep the protein cold throughout the

pHisdependent
ontemperature
anddilution
Inexpensive
Tris

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.

purification. Typically, purification is performed at 4C,

as this lower temperature both slows down the rate of
proteolysis (in the event of contaminating proteases)
andpromotesstructuralintegrityofproteins.
IntroductiontoColumnChromatography
EquipmentforColumnChromatography

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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ProteinPurification

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
https://www.labome.com/method/ProteinPurification.html

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.

Column chromatography can be performed with automated systems

(Figure2),whichuseapumptoforcesolventoverapackedcolumnat
a set flow rate, or can be run by gravity flow. Both automated and
gravity flow systems can be coupled to automatic fraction collecting
systems. There are advantages and disadvantages to each system.
GEHealthcareAKTAFPLCsystemisaverycommonchoice[514].
TypesofColumnChromatography
The four major types of column chromatography include affinity
chromatography, ion exchange chromatography (IEX), hydrophobic
interactionchromatography(HIC),andsizeexclusionchromatography
(SEC).Mostpurificationschemesrequiretheuseofmorethanoneof
these
types
of
chromatographic procedures

TypeofSystem

Advantages

to yield the necessary purity

for downstream applications.
Choosing
the
most

Disadvantages

Canletrunbyitself
Oftencoupledtoan
absorbancedetector
Automated

Canprogram
equilibrationand
washsteps
Easytosetupa
gradientforelution

Requiresspecific,
costlyequipment
Maximumflowrate
isdependenton

these methods is essential in

optimizing
a
protein
purificationscheme.

pressurelimitof
column

By analyzing the sequence of

a
protein,
unique
characteristics
can
be
identified that might assist in

Veryreproducible
Lessexpensive
Theuserhasmore
control

GravityFlow

Canmake
adjustmentsduring
run

appropriate chromatographic
method(s) and the order of

Morelaborintensive
Flowroteislimited
bygravity

its purification. The size and

charge of a protein (at a
specific
pH)
can
be
determined, along with the
identificationoflargestretches
ofhydrophobicresidues.
AffinityChromatography

Table3.Systemsforthechromatographicseparationofproteins.
SummarizedfromGE.

Affinity chromatography relies

on the specific and reversible

binding of a protein to a
matrixbound ligand. The
Typeof
Chromatography

ligand can either bind directly

to the protein of interest or to
a tag that is covalently
attachedtotheprotein.Affinity
chromatography is often the
most

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)

procedure and is typically

used in early stages of the
purification
scheme.
Depending
downstream

on
the
application,

affinity purification might be

theonlychromatographicstep
required to achieve adequate
purity.
The stationary phase for
affinity chromatography is

IonExchange

Netsurfacecharge

Lowionicstrength

Highionicstrength
Increased(cation
exchange)or
decreased(anion
exchange)pH

Hydrophobic
Interaction

Hydrophobicity

Highionicstrength

Lowionicstrength

SizeExclusion

Hydrodynamic
radius

Table4.Thefourmostcommontypesofcolumnchromatographyusedin
proteinpurification.

made of an inert matrix

covalentlyattachedtoaligandthatspecificallybindsto
a protein or group of proteins. The inert matrix is
typically composed of crosslinked agarose or
polyacrylamide. Proteins can be purified by affinity
chromatographyinaselectiveornonselectivemanner.
Inselectiveaffinitychromatography,aligandspecificfor
a protein or a covalently attached tag is used. In non
selectiveaffinitychromatographysuchasProteinA,G,
L for immunoglobulin, or heparin for DNAbinding
proteins, or lectin for glycoproteins, the ligand binds to
agroupofproteinswithsimilarbindingcapabilities.
In both types of affinity chromatography, proteins are
loaded on the column under conditions that influence
bindingbetweentheprotein(ortag)anditsligand.The
bound protein is washed under conditions that do not
disruptthespecificinteraction,butthatcandisruptany
nonspecific interactions between contaminating

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

known structures) to assist in purification. See

Labome survey on protein/protide tags for a list of

Imidazoleor

https://www.labome.com/method/ProteinPurification.html

(or in rare cases, in flexible loops of proteins with

commontagsandtagrelateddiscussions.
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ProteinPurification

taggedprotein

Ni2+orCo2+

freehistidine

FLAGtagged
protein

FLAG
specific
antibody

FLAG
peptideor
lowpH

specific interaction that occurs between an antibody

and its antigen (the sequence recognized by the

GSTtagged
protein

Reduced
glutathione

Free
glutathione

coupled to a matrix for specific binding of the

Myctagged
protein

Mycspecific
antibody

LowpH

Antibody
(classspecific)

ProteinA,
G,orL

Extremesin
pH

DNAbinding
protein

Heparin

Highionic
strength

Antibodies are often purified based on the highly

antibody). A peptide containing the antigen can be

antibody. Lowering the pH of the elution buffer
disrupts the antibody/peptide interaction to release
the bound antibody. This method is often used
commerciallyintheisolationofantibodiesfromcrude
serum.
Proteins
can also

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.

https://www.labome.com/method/ProteinPurification.html

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IonExchangeChromatography
(IEX)

Problem

Proteindoesnotbind

charge, through electrostatic

interactions
that
occur
between

proteins

and

charged stationary phase.

Two types of IEX exist: (1)
anion exchange (positively

Proteindoesnotelute

charged stationary phase that

binds to negatively charged
proteins)

and

(2)

cation

exchange (negatively charged

stationary phase that binds to
positively charged proteins).
Ionexchangechromatography

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

charge that depends on the

aminoacidcompositionofthe
protein and any covalently

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
https://www.labome.com/method/ProteinPurification.html

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structure of the protein into consideration. In its native state, some

residues of a protein are more exposed than others and, thus, the
actual pI and net surface charge is sometimes different from those
determinedtheoretically[21].Thedistributionofaminoacidsrelative
to one another can also affect a protein's pI [22], and this is not
taken into consideration with theoretical pI calculations. Some
techniquesallowfortheexperimentaldeterminationofaprotein'spI
initsnativestate,includingelectrophoreticisoelectricfocusing[23],
capillary isoelectric focusing [24], and a more recent high
throughputluminescencebasedapproach[25].
Typically,bindingofaproteintoIEXmustbedeterminedbytrialand
error, using solvents with a range of pH values, to determine the
optimumpHforproteinretention.AsolventpHthatisaboutonepH
unit away from the pI is usually sufficient for protein binding [26]
however,insomecasesapHfurtherfromthepIisrequired[27].
ThestationaryphaseofIEXiscomposedofaninertagarosebased
or polymeric matrix with a covalently bound charged group. Matrix
particlesareavailableinmanysizes,andcaneitherbenonporous
orcancontainporesofvariablesize.Thechoiceofmatrixdepends
on the desired binding capacity, resolution, and flow rate. Smaller
particlesizesofferhigherresolution,butrequirelowerflowratesand
take longer to run. Porous matrices offer a higher binding capacity
than nonporous matrices. In a nonporous matrix, proteins cannot
enter the resin and, thus, offer increased sample recovery, greater
resolution, and shorter run times than porous matrices. One study
showed that, for most proteins, resolution and recovery are similar
when separated by a porous versus nonporous matrix however,

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)
https://www.labome.com/method/ProteinPurification.html

2.carboxymethyl(CM)weakcationexchanger

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gradient.Fractionswereanalyzedforboth
totalproteincontentandactivityspecific
totheproteinofinterest.Thepeak
centeredatfraction45containsthe
proteinofinterest,asindicatedbyprotein
activity.From
http://www.insectscience.org/9.04/.

Strong exchangers should be used when the pH required for

binding is very acidic or basic (assuming this pH is also
appropriate for maintaining protein stability), as the functional
groups remain charged over a larger pH range, [29]. Weak
exchangers have been shown to provide less retention of
some proteins, enabling their elution at lower ionic strength

[30]. Because high ionic strength can affect the stability of

someproteins[31],weakexchangersmightbemoresuitableforproteinsthatdonotrequireanextremepHfor
binding.
Inionexchangechromatography,thestationaryphaseisfirstequilibratedwithalowionicstrengthbuffer.The
protein sample is then loaded onto the stationary phase in the same low ionic strength buffer used for
equilibration.Theboundproteiniswashedextensivelybeforeelutionwithahigherionicstrengthbufferor,in
some cases, a buffer with altered pH (Figure 6). Counterions in the elution buffer interact with the charged
stationary phase, displacing the bound protein. Certain salts are more efficient for use in ion exchange
chromatographythanothers,duetotheirabilitytodisplaceboundproteinsandtheireffectsonproteinstability
[32].TypicallyNaClorKClisusedforelution,withNa+orK+servingasthecountercationincationexchange
chromatographyandClservingasthecounteranioninanionexchangechromatography.Alternatively,thepH
ofthebuffercanbealteredtoreducetheprotein'schargeanddisruptitsinteractionwiththestationaryphase.
Forproteinsboundtoacationexchangestationaryphase,increasingthebufferpHwillleadtoalesserpositive
charge on the protein, and subsequent elution from the column. For proteins bound to an anion exchange
stationaryphase,decreasingthebufferpHwillleadtoalessernegativechargeontheprotein,andsubsequent
elutionfromthecolumn.
Alternatively, the pH of the buffer can be adjusted so
that the protein of interest does not bind to the ion
exchange stationary phase while contaminating
proteins do. In this case, the protein of interest is
collectedintheflowthrough,whilesomecontaminating
proteinsareremovedbytheirbindingtothestationary
phase.
As with other chromatographic methods, ion exchange
chromatography may require some troubleshooting to
determine the optimum conditions for protein binding,
proteinelution,andadequateresolution.
HydrophobicInteractionChromatography(HIC)

Hydrophobic interaction chromatography (HIC)

separates proteins based on their hydrophobicity, and
is often used as an intermediate step in a purification
scheme.Proteinsareboundtoastationaryphaseina

Figure9.Amixtureofthreeproteinsofvarying
hydrodynamicradiusisloadedontoasize
exclusioncolumn.Largeproteinselutefirst,as
theyareunabletoentertheporesofthematrix
andhaveastraightforwardpaththroughthe
column.Smallerproteinscanenterthepores,
haveamoreconvolutedpathand,thus,take
longertotraversethematrixandelutefromthe
column.
high ionic strength buffer and,

Problem

Cause

Solution

thus, HIC can typically be

performed immediately after

Runmoreequilibration
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ProteinPurification

Columnwasnot
equilibrated

bufferthroughcolumnand
reloadprotein

ionexchangechromatography

Ionicstrengthofbinding
bufferistoohigh

Lowerionicstrengthof
buffer
AdjustbufferpH(lowerfor
cationexchange,higherfor
anionexchange)

dilution required. HIC is also

commonly performed after an

pHisnotfarenoughfrom
pI

Proteindoesnotelute

Lowresolution

with no buffer exchange or

ammonium

sulfate

precipitation, a procedure that

canbeusedtoquicklyremove

Ionicstrengthofelution
bufferistoolow

Increaseionicstrength

Proteinaggregatedon
column

Adjustbufferconditionsfor
moreproteinstability

Flowrateiseithertoofast
ortooslow

Adjustflowrate

Columnwasnotwashed
sufficiently

Washwithahigherionic
strengthbufferClean
stationaryphaseaccording
tomanufacturer

Proteinaggregatedon
column

Adjustbufferconditionsfor
moreproteinstability

The presence of salt ions in

Proteinisunfoldedor
aggregated

Adjustbufferconditionsfor
moreproteinstability

solution can lead to partial

unfolding of a protein and the

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.

exposure of normally buried

Addcofactor

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

molecule. Unlike the other

chromatographic procedures
described previously, proteins
do not bind to a stationary
phase in SEC. Instead,
proteins are separated by the

Proteindoesnotelute

speed at which they navigate

through an inert stationary
phase(Figure9).
SEC is a valuable method
most often used in the final
stepsofapurificationscheme

[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

species [36] can all be

separated from the native
protein, while simultaneously

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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Superdex is often the best

choice for typical SEC
procedures,asitiscompatible
with most solvents and offers
thehighestresolutionofallthe
availablestationaryphases.
Aswithotherchromatographic
methods,
there
are
advantages
and
disadvantages to SEC, and
the decision to use this
method depends on both the
protein and its downstream
application.
AdvantagesofSEC:

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.

achieve maximum resolution

is often timeconsuming, but
certain factors can have a
hugeimpactonresolution.
Conditions that increase
proteinresolutionwithSEC:

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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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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