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StudyingProteinInteractionsbyFar-WesternBlotting

Far-Westernblottingwasoriginallydevelopedtoscreenproteinexpressionlibrarieswith³²P-labeledglutathioneS-transferase(GST)-fusionprotein.Far-Westernblottingisnowusedtoidentifyprotein:proteininteractions.Inrecentyears,far-Westernblottinghasbeenusedtodeterminereceptor:ligandinteractionsandtoscreenlibrariesforinteractingproteins.WiththismethodofanalysisitispossIBLetostudytheeffectofpost-translationalmodificationsonprotein:proteininteractions,examineinteractionsequencesusingsyntheticpeptidesasprobes,andidentifyprotein:proteininteractionswithoutusingantigen-specificantibodies.Far-WesternBlottingvs.WesternBlottingThefar-WesternblottingtechniqueisquitesimilartoWesternblotting.InaWesternblot,anantibodyisusedtodetectthecorrespondingantigenonamembrane.Inaclassicalfar-Westernanalysis,alabeledorantibody-detectable“bait”proteinisusedtoprobeanddetectthetarget“prey”proteinonthemembrane.Thesample(usuallyalysate)containingtheunknownpreyproteinisseparatedbySDS-PAGE(sodiumdodecylsulfate-polyacrylamidegelelectrophoresis)ornativePAGEandthentransferredtoamembrane.Whenattachedtothesurfaceofthemembrane,thepreyproteinbecomesaccessibletoprobing.Aftertransfer,themembraneisblockedandthenprobedwithaknownbaitprotein,whichusuallyisappliedinpureform.Followingreactionofthebaitproteinwiththepreyprotein,adetectionsystemspecificforthebaitproteinisusedtoidentifythecorrespondingband(Table4).

Table4.ComparisonofWesternBlottingandfar-WesternBlottingMethods
Step	WesternBlotting	Far-WesternAnalysis
GelElectrophoresis	NativeorDenaturing(usually)	Native(usually)orDenaturing
TransferSystem	Optimalmembraneandtransfersystemdeterminedempirically	Optimalmembraneandtransfersystemdeterminedempirically
BlockingBuffer	Optimalblockingsystemdeterminedempirically	Optimalblockingsystemdeterminedempirically
Detection(severalpossiblestrategies)*	Unlabeledprimaryantibody.–>Enzyme-labeledsecondaryantibody.–>SubstrateReagent	Unlabeledbaitprotein.–>Enzyme-labeledbait-specificantibody.–>SubstrateReagent
	Enzyme-labeledprimaryantibody.–>SubstrateReagent	RADIolabeledbaitprotein.–>Exposuretofilm
[Arrowsdesignatesequenceofstepsofdetectionstrategy]	Biotinylatedantibody.–>Enzyme-labeledstreptavidin.–>SubstrateReagent	Biotinylatedbaitprotein.–>Enzyme-labeledstreptavidin.–>SubstrateReagent
		Fusion-taggedbaitprotein.–>Tag-specificantibody.–>Enzyme-labeledsecondaryantibody.–>SubstrateReagent
*Labeledantibodiesgenerallyareenzyme-labeled(eitherhorseradishperoxidaseoralkalinephosphatase).Bycontrast,baitproteinsgenerallyarenotenzyme-labeledsincealargeenzymelabelislikelytostericallyhinderunknownbindingsitesbetweenbaitandpreyproteins.Otherlabelinganddetectionschemesarepossible.

Specializedfar-WesternAnalysisBycreativedesignofbaitproteinvariantsandothercontrols,thefar-Westernblottingmethodcanbeadaptedtoyieldveryspecificinformationaboutprotein:proteininteractions.Forexample,Burgess,etal.usedamodifiedfar-Westernblottingapproachtodeterminesitesofcontactamongsubunitsofamulti-subunitcomplex.Byan“orderedfragmentladder”far-Westernanalysis,theywereabletoidentifytheinteractiondomainsofE.coliRNApolymeraseßsubunit.Theproteinwasexpressedasapolyhistidine-taggedfusion,thenpartiallycleavedandpurifiedusingaNi²⁺-chelateaffinitycolumn.Thepolyhistidine-taggedfragmentswereseparatedbySDS-PAGEandtransferredtoanitrocellulosemembrane.Thefragment-localizedinteractiondomainwasidentifiedusinga³²P-labeledproteinprobe.ImportanceofNativePreyProteinStructureinfar-WesternAnalysisFar-Westernblottingproceduresmustbeperformedwithcareandattentiontopreservingasmuchaspossiblethenativeconformationandinteractionconditionsfortheproteinsunderstudy.Denaturedproteinsmaynotbeabletointeract,resultinginafailuretoidentifyaninteraction.Alternatively,proteinspresentedinnon-nativeconformationsmayinteractinnovel,artificialways,resultingin“falsepositive”interactions.Thepreyproteininparticularissubjectedtopreparativeprocessingstepsforfar-Westernblottingthatcanhavesignificanteffectsondetectionofprotein:proteininteractions.Thisisnottoimplythatidentificationofvalidinteractionsisnotpossiblebutonlytostresstheimportanceofappropriatevalidationanduseofcontrols.CriticalStepsinFar-WesternAnalysisGelElectrophoresisSeparationofproteinsbySDS-PAGE(i.e.,denaturingconditionswithorwithoutareducingagent)offersmoreinformationaboutMW,presenceofdisulfidesandsubunitcompositionofapreyprotein,butmayrenderthepreyproteinunrecognizablebythebaitprotein.Inthesecases,theproteinsmayneedtobesubjectedtoelectrophoresisundernativeconditions,i.e.,nondenaturingandwithoutreducingagent.TransfertoMembraneAfterseparationonthegel,proteinsareelectrophoreticallytransferredfromthegeltoamembranein2-16hours.Thetypeofmembrane(e.g.,nitrocelluloseorPVDF)usedforthetransferofproteinsiscritical,assomeproteinsbindselectivelyorpreferablytoaparticularmembrane.Theefficiencyandrateofproteintransferisinverselyproportionaltothemolecularweightoftheprotein.Insomecases,transferconditionsaltertheshapeoftheproteinanddestroyorstericallyhindertheinteractionsiteontheprotein.Forfar-Westernanalysis,itisessentialthatatleasttheinteractiondomainofthepreyproteinisnotdisruptedbythetransferorisabletore-foldonthemembranetoformathree-dimensional(3-D)structurecomprisinganintactinteractionsite.Generally,asignificantpercentageoftheproteinpopulationrenaturesuponremovalofSDS.WhenSDSiseliminatedduringthetransferprocess,transferredproteinsgenerallyrenaturewithgreaterefficiencyandarethereforemoreeasilydetectedbyfar-Westernblotting.Intheeventthattheproteinisunabletore-foldtocreateanintactbindingsite,itmaybenecessarytoaddadenaturation/renaturationsteptotheprocedureortoperformtheprotein:proteininteractionin-gelwithouttransfer(SeeIn-Gelfar-WesternDetectionbelow).Denaturation/renaturationistypicallyaccomplishedusingguanidiniumhydrochloride.BlockingBufferAftertransferringproteinstothemembrane,Westernblottingproceduresrequirethatunreactedbindingsitesonthemembranebeblockedwithanon-relevantproteinsolution.Inadditiontoblockingallremainingbindingsitesonthemembrane,ablockingbufferreducesnonspecificbindingandaidsinproteinrenaturationduringtheprobingprocedure.Avarietyofdifferentproteinblockersmaybeused,andnooneblockingproteinsolutionwillworkforallblottingexperiments.Anygivenproteinblockermaycross-reactorotherwisedisruptthespecificprobinginteractionbeingstudied.Determinationofaneffectiveblockingbuffermustbemadeempirically.Often,bovineserumalbumin(BSA)isusedasastartingpointformanymembrane-probingreactions.Insufficientblockingmayresultinhighbackground,whereasprolongedblockingcouldresultinaweakormaskedsignal.Renaturationoftheproteinalsoappearstooccurduringtheblockingstepsoitisimportanttooptimizetheblockingconditionstoobtainthebestsignal-to-noiseratioforeachapplicationandthennotdeviatefromthemethod.BindingandWashConditionsProtein:proteininteractionsvarydependingonthenatureoftheinteractingproteins.ThestrengthoftheinteractionsmaydependonthepH,saltconcentrationsandthepresenceofcertainco-factorsduringincubationwiththebaitprotein.Someprotein:proteininteractionsmayalsorequirethepresenceofadditionalproteins.Whateverthenecessaryconditions,theywillneedtobemaintainedthroughouttheproceduretomaintaintheinteractionuntilitcanbedetected.Thismayinfluencetheformulationofwashingbufferusedbetweenprobingsteps.DetectionMethodsDependingonthepresenceofalabelortagonthebaitprotein,oneoffourdetectionmethodsisusedtodetectfar-Westernblotprotein:proteininteractions:

Directdetectionofpreyproteinwitharadioactivebaitprotein
Indirectdetectionwithantibodytothebaitprotein
Indirectdetectionwithantibodytothetagofafusion-taggedbaitprotein
Detectionwithbiotinylatedbaitproteinandenzyme(HRP/AP)labeledwithavidinorstreptavidin

Eachmethodhasitsownadvantagesanddisadvantages.Severalmethodsareusedtogenerateradioactiveisotopelabelsonbaitproteins.Theisotope³²Piscommonlyusedtolabelfusion-taggedproteinprobesatphosphorylationsitesonthetag.Thismethodofphosphorylationhaslittleeffectontheprotein:proteininteractionbecausethephosphorylationsiteislocatedinthefusiontagportionoftheprotein.Anotherradioactivemethodinvolvesdirectlabelingofbaitproteinusingendogenousphosphorylationsites.However,thistechniquecanonlybeusedif³²Plabelingofthesesitesdoesnotinterferewithprotein:proteininteractions.6Radioactivedetectionhasalsobeenusedwhenprobesaremadebyincorporationof³⁵S-methionineduringinvitrotranslation.Onedisadvantageofthismethodisthatitcanonlybeusedforproteinprobesthathavemultiplemethionineorcysteineresidues.AlthoughradioactiveisotopesgenerallydonotinterferewithinteractionsbecausetheyonlyalterMWbyafewatomicmassunits,isotopicdetectionmethodshaveseveraldisadvantagesincludinghealthhazardsanddisposalissues.GST-taggedorpolyhistidine-taggedrecombinantlyexpressedbaitproteinsareoftendetectedwithaprimaryorenzyme-labeledantibodyspecifictothetag.Antibodiestoboththesepopularfusiontagsarecommerciallyavailable.Whenrecombinanttechniquescannotbeusedtocreatefusiontaggedbaitproteinsandbait-specificantibodiesarenotavailable,baitproteinscanbebiotinylatedanddetectedwithlabeledavidinorstreptavidin.Pierceoffersafulllineofbiotinylationreagentsandenzyme-labeledavidinandstreptavidin(refertotheProteinDetectionsection).Althoughlysatecontainingthebaitproteincanbeusedforprobingmembranes,thiscanresultinhighbackground(lowsignal-to-noise);therefore,itispreferabletopurifythebaitproteinbeforeprobing.Whateverthemethodofnon-isotopiclabelingused,thelastprobingstepusuallyinvolvesuseofanantibodyorstreptavidinprobethatisconjugated(labeled)withanenzymewhoselocalizedactivityonthemembranecanbedetectedbyincubationwithasuitablecolorimetric,chemiluminescentorfluorogenicsubstrate.Horseradishperoxidase(HRP)andalkalinephosphatase(AP)arethemostpopularenzymelabelsusedforthispurpose,withHRPbeingthemostversatile.AswithtraditionalWesternblotting,sensitivityinfar-Westernblottingdependslargelyontheenzymesubstratesystemusedfordetection.PatentedSuperSignalChemiluminescentSubstrateTechnologyenablesunmatchedsensitivityandlowerlimitsofdetectionforHRP-basedconjugate.ControlsWhenidentifyingprotein:proteininteractionsbythefar-Westerntechnique,itisimportanttoalwaysincludeappropriatecontrolstodistinguishtrueprotein:proteininteractionbandsfromnonspecificartifactualones.Forexample,experimentsinvolvingdetectionwithrecombinantGSTfusionproteinsshouldbereplicatedwithGSTalone.Abaitproteinwithamutationinthepredictedinteractiondomaincanbeprocessedasacontroltodeterminespecificityoftheprotein:proteininteraction.Anon-relevantproteincanbeprocessedalongsidethepreyproteinsampletoactasanegativecontrol.Ideally,thecontrolproteinwouldbeofsimilarsizeandchargetotheproteinunderinvestigationandwouldnotinteractnonspecificallywiththebaitprotein.Inapproachesthatuseasecondarysystemfordetectionofthepreyprotein,suchasenzyme-labeledstreptavidinwithabiotinylatedbaitprotein,itisimportanttoincludeaduplicatecontrolmembranethatisprobedonlywiththelabeledstreptavidin.Thiswouldrevealanybandsresultingfromendogenousbiotininthesampleornonspecificbindingofthelabeledstreptavidin.Whenafusiontagisusedwithacorrespondingantibody,itiscriticaltoprobeoneofthecontrolmembraneswiththelabeledantibodyalone.Thiscontrolhelpstoconfirmthattherelevantbandisnotduetononspecificbindingofthelabeledsecondaryantibody.Toobtainmeaningfulresults,appropriatetestandcontrolexperimentsshouldbesubjectedtogelelectrophoresis,transferandprobinginparallel.(backtotop)In-Gelfar-WesternDetection*AdvantagesofIn-GelDetectionBecauseofrestrictionsassociatedwiththetransferprocess,blocking,andthepossibilityofnonspecificbindingofbaitproteinstounrelatedbandsonthemembranes,itissometimesadvantageoustoperformfar-Westerndetectionwithinthegel.Inthisprocedurepreyproteinsamplesareseparatedinprecastgelsusingeithernativeordenaturingconditions.Followingelectrophoresis,thegelsarepre-treatedwith50%isopropylalcoholandwatertoremoveSDSfromthegelandallowthepreyproteintorenature.Thegelisthenincubatedwiththebaitprotein(usuallyinthepureform).Ifthebaitproteinisbiotinylated,itissubsequentlydetectedwithstreptavidin-HRPandahighlysensitiveformulationofPierce’spatentedSuperSignalChemiluminescentSubstrate.Ifthebaitproteinisfusion-tagged,detectioniswithananti-tagHRP-conjugatedantibodyandthechemiluminescentsubstrate.Thesamecontrolsandexperimentalconditionsnecessaryforoptimizationofmembrane-basedfar-Westernsapplytoin-geldetection.Within-geldetectiontheblockingstepcanbeeliminatedbutthe“bait”proteinandthelabeleddetectionproteinmustbedilutedintheblockingbuffertoreducenonspecificbinding.Also,higheramountsofpreyandbaitproteinsareoftenrequiredfordetectioncomparedtomembranedetectionwiththeequivalentchemiluminescentsubstrate.ProFoundFar-WesternProtein:ProteinInteractionKitsPierceprovidestwokitsforfar-Westernanalysis.Thesekitsareoptimizedfordetectionbothonmembraneorin-gel.Onekitallowsthedetectionofbiotinylatedbaitproteins(Product#23500)andtheotherallowsforthedetectionofGST-taggedbaitproteins(Product#23505).Bothkitsincludeblockingandwashbuffers,HRP-labeleddetectionprotein(Streptavidin-HRPorAnti-GST-HRP)andanextremelysensitiveformulationofUnBlotChemiluminescentSubstrateoptimizedforbothon-membraneandin-geluse.Figure6illustratesthegeneralstrategyforfar-Westernanalysis.Formoredetailonthesekits,refertotheproductsection.

Figure6.Generalprincipleoffar-Westernanalysis.TheProFoundFar-WesternProtein:ProteinInteractionKitsfollowthenon-radiolabeledbaitpath.

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