MATCHMAKER-Two-Hybrid-System-3

CLONTECHInnovativeToolstoAccelerateDiscoveryMATCHMAKERGAL4Two-HybridSystem3&LibrariesUserManualPT3247-1(PR94575)Published22June1999SeeListofComponentsforstorageconditionsFORRESEARCHUSEONLYMATCHMAKERGAL4Two-HybridSystem3&LibrariesUserManualCLONTECHLaboratories,Inc.:AYeastTwo-HybridScreen8III.ListsofComponents9IV.AdditionalMaterialsRequired10V.YeastStrains&Phenotypes11A.YeastHostStrains11B.Phenotypes11VI.ControlVectors13A.PositiveControls13B.NegativeControl13VII.MATCHMAKERGAL4cDNA&GenomicLibraries14A.LibraryConstruction14B.LibraryQualityControlInformation15VIII.PreparingforaTwo-HybridScreen16A.ConstructFusionGenes16B.ObtainorConstructandADFusionLibrary16C.VerifythatConstructsDoNotActivateReporterGenes17D.VerifyProteinExpression17IX.LibraryTransformation&ScreeningProtocols18A.TransformationScales18B.YeastTransformationProtocols19C.PlatingandScreeningTransformationMixtures21D.Calculations23X.Analysis&VerificationofPutativePositiveClones24A.RetestPhenotypes24B.IsolatePlasmidDNAfromYeast24C.SortColoniestoEliminateDuplicates24D.RescueAD/libraryPlasmidsviatransformationofE.coli24E.RetestProteinInteractionsinYeast25F.InvitroAnalysis26G.SequencingAD/LibraryInserts27H.InvivoAnalysis27I.AdditionalTwo-HybridTests27XI.TroubleshootingGuide28XII.References30XIII.RelatedProducts32AppendixA:Media&SolutionRecipes33AppendixB:LibraryTitering34AppendixC:PlasmidLibraryAmplification35TableofContentsMATCHMAKERGAL4Two-HybridSystem3&LibrariesUserManualProtocol#PT3247-1(Tel:[phone];Fax:[phone]).YeaststrainY187islicensedfromBaylorUniversity.AH109isthepropertyofCLONTECHLaboratories,Inc.,andisaderivativeofPJ69-2AwhichisthepropertyoftheUniversityofWisconsinResearchFoundation(WARF).pGBKT7isaderivativeofpODB-8,whichislicensedfromtheUniversitedéBordeaux.ThepBridgeThree-HybridVectoristhepropertyoftheInstitutNationaldelaSantéetdelaRechercheMédicale(INSERM).Thisproductisintendedtobeusedforresearchpurposesonly.Itisnottobeusedfordrugordiagnosticpurposesnorisitintendedforhumanuse.CLONTECHproductsmaynotberesold,modifiedforresale,orusedtomanufacturecommercialproductswithoutwrittenapprovalofCLONTECH.MATCHMAKERGAL4Two-HybridSystem3&LibrariesUserManualCLONTECHLaboratories,Inc.(DNA-BD),whileanothergeneorcDNAisexpressedasafusiontotheGAL4activationdomain(AD;Fields&Song,1989;Chienetal.,1991).Whenbaitandlibraryfusionproteinsinteract,theDNA-BDandADarebroughtintoproximity,thusactivatingtranscriptionoffourreportergenes(Figure2).Thistechnologycanbeusedtoidentifynovelproteininteractions,confirmsuspectedinteractions,anddefineinteractingdomains.Moreover,two-hybridtechnologyprovidesimmediateaccesstothegenesencodingtheinteractingproteins.SensitiveinvivoassayYeasttwo-hybridsystemsprovideasensitivemethodfordetectingrelativelyweakandtransientproteininteractions.Suchinteractionsmaynotbebiochemicallydetectable,butmaybecriticalforproperfunctioningofcomplexbiologicalsystems(Guarente,1993;Estojaketal.,1995).ThesensitivityofMATCHMAKERGAL4Two-HybridSystem3isprimarilyattributabletohigh-foldamplificationofpositivesignalsinvivo(i.e.,transcriptional,translational,andenzymatic).Inaddition,becausethetwo-hybridassayisperformedinvivo,theproteinsaremorelikelytobeintheirnativeconformations,whichmayleadtoincreasedsensitivityandaccuracyofdetection.MATCHMAKERTwo-HybridSystem3isanadvancedGAL4-basedtwo-hybridsystemthatprovidesatranscriptionalassayfordetectingproteininteractionsinvivoinyeast.Youcanusethissystemtoscreenalibraryfornovelproteinsthatinteractwithaknownbaitprotein,ortotesttwoprevious