Pull-Down-Assays

AdvancedPartofThermoFisherScientificEmailAddress:Password:ForgotPasswordRegisterWhyRegister?CartisEmptyQuickOrderOrderingInformation»Shipping&Handling»ReturnsInfo»SecurityInfo»Terms&ConditionsEmailSign-upFollowUsHomeProteinMethodsLibraryPull-DownAssaysPull-DownAssaysElucidatinggenefunctioninvolvesdeterminingthefunctionoftheencodedproteinproduct.Inthecell,proteinsparticipateinextensivenetworksofprotein-proteininteractionsthattaketheformofdynamic“proteinmachines”,whichassembleanddisassembleinconcertwithanever-changinginfluxofintra-,interandextracellularcues.Apreliminarystepinunderstandingproteinstructureandfunctionistodeterminewhichproteinsinteractwitheachother,therebyidentifyingtherelevantbiologicalpathways.Thepull-downtechniquehasbecomeaninvaluabletoolforthelifescientistinterestedinstudyingcellularpathwaysviaprotein-proteininteractions.PageContents:IntroductiontoPull-DownAssaysCriticalComponentsofPull-DownAssaysPull-DownMethodologiesReferencesOrderFreeLiterature...ProteinInteractionTechnicalHandbookIntroductiontoPull-DownAssaysThepull-downassayisaninvitromethodusedtodetermineaphysicalinteractionbetweentwoormoreproteins.Pull-downassaysareusefulforbothconfirmingtheexistenceofaprotein-proteininteractionpredictedbyotherresearchtechniques(e.g.,co-immunoprecipitation)andasaninitialscreeningassayforidentifyingpreviouslyunknownprotein-proteininteractions.Pull-downassaysareaformofaffinitypurificationandareverysimilartoimmunoprecipitation,exceptthatabaitproteinisusedinsteadofanantibody.Affinitychromatography(i.e.,affinitypurification)methodologiesgreatlyenhancethespeedandefficiencyofproteinpurificationandsimultaneouslyprovidethetechnologyplatformtoperformapull-down,orco-purification,ofpotentialbindingpartners.Inapull-downassay,abaitproteinistaggedandcapturedonanimmobilizedaffinityligandspecificforthetag,therebygeneratingasecondaryaffinitysupport’forpurifyingotherproteinsthatinteractwiththebaitprotein.Thesecondaryaffinitysupportofimmobilizedbaitisthenincubatedwithaproteinsourcethatcontainsputativepreyproteins,suchasacelllysate.Thesourceofpreyproteinatthisstepdependsonwhethertheresearcherisconfirmingapreviouslysuspectedprotein-proteininteractionoridentifyinganunknowninteraction.ThemethodofproteinelutiondependsontheaffinityligandandrangesfromusingcompetitiveanalytestolowpHorreducingbuffers.Besidesinvestigatingtheinteractionoftwoormoreproteins,pull-downassaysareapowerfultooltodetecttheactivationstatusofspecificproteins.Forexample,proteinsthatareactivatedinresponsetotyrosinephosphorylationcanbepulleddownusinganimmobilizedSH2domainthattargetsthephosphorylatedtyrosineonagivenprotein.Additionally,GTPases,whichactasmolecularswitchesthatregulatecellsignalingbycyclingbetweenaGTP-bound(active)andGDP-bound(inactive)state,canbepulleddownusinganimmobilizedGTPase-bindingdomainofdownstreamproteinsthatarerecruitedtoGTP-bound,activatedGTPases.Inbothtypesofpull-downassays,becausethespecificityoftheinteractionisdependentonthesequenceofthebindingdomain,theseapproachesarehighlyspecificindetectingtheactivationofdistinctproteins.Learnmore...OverviewofProtein-ProteinInteractionAnalysisImmunoprecipitationCo-ImmunoprecipitationViewproducts...Tag-basedpull-downkitsActiveGTPasepull-downanddetectionkitsProductsProteinMethodsProductBlogTechnicalResourcesOrderingAboutUsContactUsHomeGeneralschematicofapull-downassay.Apull-downassayisasmall-scaleaffinitypurificationtechniquesimilartoimmunoprecipitation,exceptthattheantibodyisreplacedbysomeotheraffinitysystem.Inthiscase,theaffinitysystemconsistsofaglutathioneS-transferase(GST)-,polyHis-orstreptavidin-taggedproteinorbindingdomainthatiscapturedbyglutathione-,metalchelate(cobaltornickel)-orbiotin-coatedagarosebeads,respectively.Theimmobilizedfusion-taggedproteinactsasthebaittocaptureaputativebindingpartner(i.e.,theprey).Inatypicalpull-downassay,theimmobilizedbaitproteinisincubatedwithacelllysate,andaftertheprescribedwashingsteps,thecompexesareselectivelyelutedusingcompetitiveanalytesorlowpHorreducingbuffersforin-gelorWesternblotanalysis.ThePull-DownAssayasaConfirmatoryToolTheconfirmationofpreviouslysuspectedinteractionstypicallyutilizesapreyproteinsourcethathasbeenexpressedinanartificialproteinexpressionsystem.Thisallowstheresearchertoworkwithalargerquantityoftheproteinthanistypicallyavailableunderendogenousexpressionconditionsandeliminatesconfusingresultsthatcouldarisefrominteractionofthebaitwithotherinteractingproteinspresentintheendogenoussystemthatarenotunderstudy.Proteinexpressionsystemlysates(i.e.,E.coliorbaculovirus-infectedinsectcells),invitrotranscription/translationreactions,andpreviouslypurifiedproteinsareappropriatepreyproteinsourcesforconfirmatorystudies.ThePull-DownAssayasaDiscoveryToolThediscoveryofunknowninteractionscontrastswithconfirmatorystudiesbecausetheresearchinterestliesindiscoveringnewproteinsintheendogenousenvironmentthatinteractwithagivenbaitprotein.Theendogenousenvironmentcanentailaplethoraofpossibleproteinsourcesbutisgenerallycharacterizedasacomplexproteinmixtureconside