蛋白质间相互作用研究方法--技术流（protein interaction research methods - technology flow）

蛋白质间相互作用研究方法技术流（PROTEININTERACTIONRESEARCHMETHODSTECHNOLOGYFLOW）RESEARCHMETHODSOFPROTEINPROTEININTERACTIONIDENTIFYPROTEINSTHATMAYINTERACTWITHTARGETPROTEINSTWOHYBRIDANDOTHERTWOCOMPONENTSYSTEMSSTAGE1IDENTIFICATIONOFBAITLEXAFUSIONPROTEINCONSTRUCTIONOFBAITLEXAFUSIONPROTEIN1THETARGETDNAENCODINGTHEBAITPROTEINWASCLONEDINTOTHEPOLYJUNCTIONOFTHELEXAFUSIONVECTORTOSYNTHESIZEALEXAINTHEFRAMEWORKFUSIONGENETHETERMINATIONSEQUENCEOFTHECARBOXYLENDOFTHEBAITSEQUENCEISDETERMINEDTHEPLASMIDFORMEDASPBAIT2USINGACOMBINATIONOFTHEFOLLOWINGLEXAFUSIONGENESANDLEXAOPLACZREPORTERPLASMIDS,ASERIESOFEGY48LEXAOPLEU2ISESTABLISHEDSELECTEDYEASTFORTRANSFORMATIONAPBAITPMW12ACTIVATIONASSAYBPSH174PMW12ACTIVATEDPOSITIVECONTROLCPRFHM1PMW12ACTIVATEDNEGATIVECONTROLDPBAITPJK101INHIBITIONOF/DNABINDINGASSAYEPRFHM1PJK101SUPPRESSEDPOSITIVECONTROLFPJK101ALONESUPPRESSEDNEGATIVECONTROLS3PLACEEACHCONVERSIONMIXTUREONTOASUITABLEALTERNATIVEDEFECTBOARDCMGLUURAHISFORPLASMIDCOMBINATIONSAEORCMGLUURAFORPLASMIDCOMBINATIONS,FCULTUREPLATESWERECULTUREDAT23FOR37DAYSTOSELECTPLASMIDSCONTAININGPLASMIDSYEASTCLONINGAPREPARATIONOF4TRANSFORMANTSIDENTIFICATIONOFACTIVATIONANDINHIBITORYACTIVITYANALYSISOFXGALANDLEU2PHENOTYPES5FROMEACHCONVERSIONOFAF,SELECTABOUT8CLONESWITHASTERILEFLATFACEDTOOTHPICKWITHACLEANTOUCHTOTAKEATOOTHPICKCLONECELLS，THEYAREDRAWNONFRESHCMGLUURAHISORCMGLUURAPLATES,AND1CMLONGLINESAREINCUBATEDAT30DEGCPASSTHENIGHT。6SECONDDAYSFROMTWOTOEACHOFTHEFOLLOWINGLINESTOTHEMOTHERBOARDPLATETRANSFORMAFLINEUPTOCMGLU,XGAL,URA,ANDCMGAL,XGALURATRANSLATEACLINEUPTOCMGLU,URAHISLEU,ANDCMGALURAHISLEUINCUBATETHEPLATEFOR4DAYSAT730DAYS8INHIBITIONANDACTIVATIONWEREANALYZEDAFORINHIBITORYACTIVITY,THEXGALPHENOTYPEWASOBSERVEDAT1224HONSTREAKBFORACTIVATION,THEXGALPHENOTYPEWASOBSERVEDATSTREAK1872HCOBSERVEDTHEPHENOTYPEOFLEUBETWEEN4896ANDH9BASEDONTHERESULTSOFINHIBITIONANDACTIVATIONANALYSIS,APPROPRIATECANDIDATECLONESWERESELECTEDDETECTIONOFBAITPROTEINEXPRESSION10ONTHEMOTHERBOARD,TAGCLONINGANDANALYSISOFPROTEINEXPRESSIONCLONESTHATHAVEBEENPROVEDTOBESUITABLEFOREXPRESSINGBAITWEREUSEDASBASALBACTERIACULTURE,FORLIBRARYCONVERSION11PAIRSOFEACHNEWDECOYWERECONSTRUCTED,ANDATLEASTTWOPRIMARYTRANSFORMANTSWEREANALYZEDTHEREAREALSOTWOPROTEINSTHATAREPOSITIVEFORPROTEINEXPRESSIONCONTROLTRANSFORMANTS12TRANSFER15MLCULTURESINTOACENTRIFUGETUBE,CENTRIFUGATION35MINATTHEMAXIMUMSPEEDONTHECENTRIFUGEITCANBESEENTHATTHEVOLUMEOFPRECIPITATIONIS25LCAREFULLYTILTORSUCKTHESUPERNATANT13ADD50LOF2SDSGELBUFFERTOTHECENTRIFUGETUBE,THERAPIDOSCILLATIONOFTHECENTRIFUGETUBETOSUSPENDPRECIPITATIONSTANDTHECENTRIFUGETUBESAREPLACEDONDRYICEORDRYICE/ETHANOLBATH14DIRECTTHESAMPLEFROMDRYICEOR70DEGCTO100DEGCANDBOILFOR5MIN15COOLEDTHESAMPLEONTHEICEANDCENTRIFUGEDFOR530SECONDSATTHEMAXIMUMSPEEDONTHECENTRIFUGEADD2050LSAMPLETOSDSEACHLANEINPOLYACRYLAMIDEGEL16ELECTROPHORESISANDANALYSISOFTHEPRODUCTTODETERMINEWHETHERTHEDESIREDSIZEOFTHEBAITPROTEINISEXPRESSEDATAREASONABLELEVEL17,INORDERTOPREVENTPOSSIBLEPROBLEMS,THEYEASTLYSATECONTAININGLEXAFUSIONBAITWASANALYZEDBYWESTERNBLOTTHESECONDSTAGESCREENINGANINTERACTINGCHILDTRANSFORMATIONLIBRARY1SELECTONEOFTHEPRIMARYCONTROLTRIALSINPHASE1,THESTATEBESTEXPRESSIONOFBAITPROTEINANDLEXAOPLACZREPORTERTHEYEASTCOLONIESWEREINOCULATEDIN20MLCMGLUURAHISLIQUIDMEDIUM,ANDWERECULTUREDOVERNIGHTAT302DILUTEDOVERNIGHTCULTURESOF20MLIN300MLCMGLUURAHISLIQUIDMEDIUMUPTOOD600WEREAPPROXIMATELY010015ONAROTATINGTABLE,SHAKEAT30DEGREESUNTILOD600REACHABOUT0503TRANSFERTHECULTURESTO1STERILECENTRIFUGEBOTTLESOF250ML,ROOMTEMPERATURE10001500GUSINGSORVALLGSAROTOR25003000R/MIN,CENTRIFUGE5MINREMOVETHESUPERNATANTANDADD30MLOFSTERILEWATERTOTHETABLELIGHTPATTHECENTRIFUGEBOTTLE,SUSPENDTHESEDIMENT,ANDTRANSFERTHEMIXTURETO150MLSTERILEFALCONTUBES410001500GIBIDCENTRIFUGALYEASTCELLS5MINDRAINWATERANDRESUSPENDYEASTCELLSAT15MLCONTAINING01MOL/LLITHIUMACETATEINTEPH7551GDNALIBRARIESAND50GDNACARRIERSWERENEWLYADDEDTOTHE3015MLASEPTICSMALLCENTRIFUGETUBESIMMEDIATELYADD50LYEASTSUSPENSIONTOEACHSMALLCENTRIFUGETUBE6STERILETEPH75CONTAINING40PEG4000AND01MOL/LLITHIUMACETATEWASADDEDTOEACHTUBEOFCELLSUSPENSION,MIXEDNOTOSCILLATING,ANDCULTUREDAT30LFOR3060MIN3007ADD40LDMSOINEACHTESTTUBE,TURNTHEBLENDINGSUSPENSION,ANDHEAT10MINONTHEHEATINGBLOCKAT42DEGC8CONVERTMIXTUREPLATES28OFTHESETUBESAREUSEDTOPRODUCETRANSFORMANTSADDEACHTUBEMIXTURETOTHE24CM24CMCMGLUURAHISTRPTHECELLSWERECOATEDEVENLYONTHEPLATE,ANDTHEPLATESWEREINCUBATEDAT30DEGCUNTILTHECOLONIESAPPEAREDTHEREMAINING2PERTUBE350LMIXTUREWASADDEDTOTHE24CM24CMCMGLUURAHISTRPPLATE,30CCULTUREPLATETOABSORBTHEREMAININGCOLONIESEACHTUBE40LMIXTUREWITHSTERILETEPH75ORWATERASATHESERIESWEREDILUTEDAT110,EACHDILUTIONTAKEN100L,TAKENONTHE100MMCMGLUURAHISTRPPLATE,ANDCULTUREDAT30DEGCTOTHECOLONIESHARVESTANDENRICHMENTOFPRIMARYTRANSFORMANTS9HARVESTTHELIBRARYBYSHAKINGORSCRAPING10IFNEEDED,ADDSTERILETEPH75ORSTERILEWATERTOACONEOF4045MLINEACHCONEFILLEDWITHYEASTCELLSANDOSCILLATEORTURNTHETESTTUBESUSPENSIONCELLS11USEATABLECENTRIFUGE10001500GATROOMTEMPERATURECENTRIFUGETUBE5MIN,DISCARDTHESUPERNATANT12REPEATSTEPS10AND1113,THESUSPENSIONOFCOMPRESSEDCELLS,SEDIMENTATIONINADOUBLEVOLUMEOFSTERILEGLYCERINSOLUTION,COMBINEDWITHDIFFERENTCONTENTSOFTHETUBE,THOROUGHLYMIXING14,RESPECTIVELY,TRANSFERRED1MLCELLMIXTUREINASERIESOFSTERILESMALLCENTRIFUGETUBES,STOREDAT70DEGREESCSCREENINGOFINTERACTINGPROTEINS15DEFROSTATRANSFORMEDLIBRARYOFYEASTCELLSFROMSTEP14USINGCMGALRAFFURATRPMEDIUM,PRESS110GAL14HWASINDUCEDBYSHAKINGCULTUREYEASTCELLSAT30DEGCTOINDUCETRANSCRIPTIONOFTHEPROMOTERINTHELIBRARY16,106CELLSWERECULTUREDONANAPPROPRIATENUMBEROF100MMCMGALRAFFURATRPLEUDROPOUTPLATESTHEPLATESWERECULTUREDAT1730DEGCFOR5DAYS18OBSERVETHEGROWTHOFPLATESANDLABELTHEMWHENTHEYARECLONED19,FIFTHDAYS,FORMINGAMOTHERBOARDTHATISGROUPEDBYDIFFERENTCLONESTHATAPPEAREVERYDAYINCUBATETHEPLATESAT2030DEGCUNTILSPOTS/COLONIESFORMFIRSTDETERMINATIONOFPOSITIVEINTERACTIONSDETECTIONOFBETAGALACTOSIDASEACTIVITYANDLEUCINEDEMAND21TRANSCRIPTIONALACTIVITYWASMEASURED22INTERPRETTHERESULTSTHETHIRDSTAGEPOSITIVEINTERACTIONISRECONFIRMEDISOLATIONOFPOSITIVEPLASMIDS1CELLLYSATESWEREPREPAREDFROMTHEPOSITIVECOLONIESTHECELLSWERECLEAVEDINSDSWHENTHEYSEPARATEDASMALLNUMBEROFCOLONIES,ANDALARGENUMBEROFISOLATESWEREISOLATEDWHENBACTERIAARECOLONIES,THECELLSARECLEAVEDWITHENZYMEHYDROLYSISENZYMESCONVERTINTOESCHERICHIACOLI2TOINTRODUCETHE15LPLASMIDDNAINTOTHEESCHERICHIACOLIDH5ALPHAORKC8INESCHERICHIACOLIBYMEANSOFELECTROPORATION,THEBACTERIAWEREPLACEDONALBAGARPLATECONTAINING50G/MLAMPICILLINANDINCUBATEDOVERNIGHTAT373IFPLASMIDDNAISTRANSFERREDINTODH5ALPHA,PROCEEDTOSTEP4,ANDIFTRANSFERREDTOKC8,THENINLB/AMPICILLINBOTTLEPLATEINCUBATEORREPLICATEPLATESTOTRANSFERCOLONIESTOBACTERIAANDINCUBATEOVERNIGHTWITHBASALCULTURESAT37THEISOLATEDCOLONIESWEREPREPAREDWITHSMALLAMOUNTSOFDNA,USINGTHESEDNATOCONVERTBACTERIALDH5ALPHACELLSASDESCRIBEDINSTEP24SMALLAMOUNTSOFDNAWEREPREPAREDFROMDH5ALPHACELLSCARRYINGLIBRARYPLASMIDS5RESTRICTIONANALYSIS,IDENTIFICATION,ORVALIDATIONOFREPEATEDSAMPLESPREPAREDWITHPOSITIVECLONESCONTAININGTHESAMEINSERTEDFRAGMENTSDIDYOUREPEATASMALLAMOUNTOFCDNASSECONDCONFIRMEDPOSITIVEINTERACTIONSREPEATEDPHENOTYPESANDSPECIFICTESTS6USINGTHEFOLLOWINGGROUPSOFPLASMIDSTOCONVERTYEASTSTRAINEGY48ANDSCREENCOLONIESONCMGLUURAHISPLATESA,PMW112,ANDPBAITB,PMW112,ANDPRFHM1C,PMW112,ANDANONSPECIFICDECOYPROTEINTHECONVERTEDYEASTOBTAINEDBYSTEP6CANBEUSED72TO3DAYSLATERELECTROPORATIONWASOBTAINEDFROMESCHERICHIACOLIKC8ANDDH5ALPHAPLASMIDSAREINTRODUCEDINTOINDIVIDUALTRANSFORMANTSACSPREADTHEMIXTUREONTOTHECMGLUURAHISTRPPLATEINCUBATETHEPLATEAT30DEGCUNTILTHECOLONYGROWSOUT8MAKEACMGLUURAHISTRPMOTHERBOARDFOREACHLIBRARYPLASMIDTHATNEEDSTOBEDETECTED9BETAGALACTOSIDASEACTIVITYANDLEUCINEDEFICIENTTYPEAREDETECTEDINTHESECONDSTAGE,STEP21TENTHERESULTSOFTHESESPECIFICTESTSWEREANALYZED,ANDTHEPOSITIVEISOLATESWERESEQUENCEDGSTFUSIONPROTEINWASUSEDTODETECTPROTEINPROTEININTERACTIONBYFARWESTERNBLOTPREPARATIONOFRADIOLABELEDPROTEINPROBES1PREPARETHEFOLLOWINGREACTIONMIXTUREINACENTRIFUGETUBEGAMMA,32P,ATP6000,CI/MMOL5LPROTEINKINASEA1,UNIT/MULTHEGSTFUSIONPROTEINWAS13MUGONGLUTATHIONEAGAROSE2PKBUFFER,125LWATERTO25LTHEREACTIONMIXTUREWASINCUBATEDAT37DEGCFOR30MINAFTERTHE2LABELINGREACTIONISCOMPLETED,ADD200LOF1PKBUFFERTOTHECENTRIFUGETUBETOCLEANTHEAGAROSEBEADSANDTHENDISCHARGEINTHETRACETHEMACHINEWASCENTRIFUGEDAT1MINATMAXIMUMSPEEDDISCARDTHESUPERNATANTCONTAININGFREERADIOACTIVENUCLEICACIDINASUITABLEMANNERANDREPEATITCLEANONCE3CUTTHELABELEDPROTEINWITHAPROTEASE,ORUSE20MMOL/LREDUCEDGLUTATHIONEIN50MMOL/LTRISPH80TOLABELTHEGSTFUSIONPROTEINFROMTHEAGAROSEBEADSSTORETHEMARKERPROTEININTHEICEBOXONTHESAMEDAYUSE。4IFTHEMARKERPROTEINISSEPARATEDFROMTHEGSTCOMPONENTPRIORTOTHEMARKERREACTION,THESEPHADEXG50COLUMNBALANCEDONTHELABELEDPROTEINWITHA1XPKBUFFERISUSEDTOREMOVETHEFREELABELEDNUCLEICACIDSONCETHEPROBEPROTEINISSEPARATEDFROMTHEFREENUCLEICACID,ITCANBEUSEDPLACETHEMARKERPROTEININTHEICEBOXANDUSEITONTHESAMEDAYPROBEFILM5THEPROTEINWASTRANSFERREDTOTHEMEMBRANEBYSTANDARDTECHNIQUETOPREPARETHEMEMBRANETOBEDETECTEDSIXCOMPLETELYCOVERTHEMEMBRANEWITHANALKALINEBUFFERANDSHAKEGENTLYAT4DEGCTOCLEANTHE10MIN7DISCARDTHEALKALINEBUFFER,COMPLETELYCOVERTHEMEMBRANEWITHACLOSEDBUFFER,ANDOSCILLATEGENTLYAT4DEGCFOR4HOVERNIGHT8ADDASTORAGEPROBEOF13GSTEP3,4TOANADEQUATEINTERACTIONBUFFERSOTHATTHEFINALCONCENTRATIONIS15NMOL/L,PREPARATIONOFLABELEDPROTEINSOLUTIONTRANSFERTHEFILMTOAPLATECONTAININGADILUTIONPROBETOCONFIRMTHEINTEGRITYOFTHEPROBESOLUTIONANDTHEMEMBRANEUNIFORMSURFACECONTACTINCUBATEDAT4DEGCFOR45H9DISCARDRADIOACTIVEPROBESOLUTIONSINASUITABLEMANNERCOMPLETEWITHPHOSPHATEBUFFEREDSALINESOLUTIONCONTAINING02TRITONX100THEFILMWASCOVEREDANDLIGHTLYOSCILLATORYAT4DEGC,INCUBATEDFOR10MINREPEATWASHING3TIMES10COMPLETELYCOVEREDTHEMEMBRANEWITHAPHOSPHATEBUFFEREDSALINESOLUTIONCONTAINING02TRITONX100AND100MMOL/LKCLANDGENTLYOSCILLATINGAT4DEGCFORWASHING10MINREPEATWASHING1TIMES11WRAPTHEFILMCAREFULLYWITHPLASTICWRAPANDEXPOSETOTHEXRAYPHYSIOLOGICALCONFIRMATIONANDEXPLORATIONOFINTERACTIONTHEBINDINGPROTEINWASDETERMINEDBYIMMUNOPRECIPITATION1WASHEDTHEAPPROPRIATECELLSON30PLATESOF10CMWITHPHOSPHATEBUFFERSCRAPETHECELLSONEACHBOARDTO1MLCOLDEBCLYSISBUFFER2TRANSFERTHESUSPENSIONOFEACHMILLILITERCELLTOACENTRIFUGETUBEANDCENTRIFUGE15MINATAMAXIMUMSPEEDOF4DEGONAMICROCENTRIFUGE3COLLECTSUPERNATANTABOUT30MLANDADD30GOFAPPROPRIATEANTIBODY,ANDSHAKETHEPRECIPITATE1HAT44ADD09MLPROTEINASEPHAROSESUSPENSION,AT4,THEIMMUNEPRECIPITATEWAS30MIN5USEANETNDETERGENTASEPHAROSEMIXTURECONTAINING900MMOL/LNACLANDREPEATIT5TIMESFINALLY,WASHITWITHNETN6SUCKOUTTHELIQUIDPARTOFTHEMIXTUREADD800LOF1SDSGLUEBUFFERTOTHEBALLANDBOILFOR4MIN7SAMPLESWEREADDEDTOTHEDISCONTINUOUSDISCONTINUOUSSDSPAGEGRADIENTGEL,ANDELECTROPHORESISWASPERFORMEDOVERNIGHTATACONSTANTCURRENTOF10MA8BYCOOMASSIEBLUESTAININGTOOBSERVEPROTEINBANDS9CUTTHETARGETTAPEFROMTHEGELANDPLACEITINACENTRIFUGETUBEWASHITTWOTIMESWITH1ML50ACETONITRILE,3MINEACHTIME10DIGESTTHEPROTEININTHEGELWITHTRYPSINANDTHENELUTETHEPEPTIDE11SEPARATIONOFPEPTIDESBYNARROWPOREHIGHPERFORMANCELIQUIDCHROMATOGRAPHYTHECOLLECTEDPEPTIDESAREAUTOMATICALLYEDMANONAABI,477A,OR494AMACHINEDEGRADATIONSEQUENCINGRAPIDANALYSISOFPREVIOUSLYIDENTIFIEDINTERACTIONSANALYSISOFINTERACTINGPROTEINSBYSURFACEPLASMONRESONANCESPECTROSCOPYUSINGBIACORERAMCCOMBINESTHESURFACEOFTHECM5SENSINGCHIPVIATHEPRIMARYAMINOGROUP1EMBEDCM5SENSORCHIPMODULEINTOBIACOREINSTRUMENT2HEPESBUFFERSALTSOLUTIONOFFILTRATIONANDDEGASSINGISADOPTED3,THETUBESCONTAININGNHS,EDC,ETHANOLAMINEANDRAMFCAND20MMOL/LHCLEACH100LAREPLACEDINTHEPROPERPOSITIONOFTHEBIACOREAUTOMATICINJECTORHOLDER4PLACEANAIRPIPEONTHEBIACOREAUTOMATICSAMPLERSTAND5OPERATETHEINSTRUMENTANDFLOWTHROUGHAFLOWTANKATAFLOWRATEOF5L/MINSIXADD75LNHSTOANAIRPIPE7ADD75MULEDCINTHESAMETUBE8MIXTHEINGREDIENTSINTHETUBULESCONTAININGNHSANDEDC9INJECT35LNHS/EDCMIXTURETOACTIVATETHESURFACE10INJECTED35LRAMFCINTOTHEACTIVATEDSURFACEINCOMBINATIONWITHANTIBODIES11INJECTIONOF35LETHANOLAMINERESULTEDININACTIVATIONOFEXCESSREACTIVEGROUPS12RAPIDINJECTIONOF10LMMOL/L20HCL,FOLLOWEDBYEXTRACLEANREMOVALOFNONCOVALENTBINDINGMATERIAL13BYPLACINGABASELINEREPORTPOINTBEFORETHEINJECTIONOFRAMC,ANDPLACINGSECONDREPORTSAT2MINAFTER20MMOL/LHCL,THEBINDINGLEVELSOFRAMCWEREMEASURED14TURNOFFTHEFLOWFLUID,CLOSETHECOMMANDWINDOW,ANDSAVETHEREPORTFILETHEBINDINGOFTHEANTITSHTOTHERAMCSURFACEWASDETECTED15STARTTHEINSTRUMENTSOTHATTHEFLOWRATEOFTHEFLOWPOOLCONTAININGRAMCIS10L/MIN16INJECTIONOF10L2G/MLANTITSH17RAPIDINJECTIONOF10L20MMOL/LHCL,ANDTHENREGENERATETHERAMCSURFACEWITHEXTRACLEAN18INORDERTODETERMINETHEREPRODUCIBILITYOFBINDINGTOTHERAMCSURFACE,THEVOLUMEREQUIREDFOR250RUSCANBEINDUCEDBYBINDINGWITHTHEANTITSHREPEATEDINJECTIONOFANTITSH19RAPIDINJECTIONOF10LMMOL/L20HCL,ANDTHENREGENERATETHERAMCSURFACEWITHEXTRACLEAN20TURNOFFTHEFLOWFLUID,CLOSETHECOMMANDWINDOW,ANDSAVETHEREPORTFILEDETECTIONOFTSHBINDINGTOCAPTUREANTITSHSURFACES21STARTTHEINSTRUMENT,THEFLOWRATEOFAFLOWPOOLCONTAININGRAMCIS10L/MIN22INJECTIONOF10L2G/MLANTITSH23INJECTIONOF25LAT200NMOL/LTSHLIMITSTHEDISSOCIATIONTIMEOF120S24RAPIDINJECTIONOF10LMMOL/L20HCL,ANDTHENREGENERATETHERAMCSURFACEWITHEXTRACLEAN25TURNOFFTHEFLOW,CLOSETHECOMMANDWINDOW,ANDSAVETHEREPORTFILEDETECTIONOFPROTEININTERACTIONSIMMUNOPRECIPITATIONCOIPSTUDYOFPROTEINPROTEININTERACTIONINCELLSGST/HISPULLDOWMPROTEINPROTEININTERACTIONINVITROFARWESTERNWESTERNWASUSEDTOANALYZETHEPROTEINTRANSFERTOTHEMEMBRANE,ORTOANALYZETHEPROTEININTERACTIONDIRECTLYONTHEGELYEASTTWOHYBRIDSYSTEMYEASTTWOHYBRIDSYSTEMISANIMPORTANTMETHODWIDELYUSEDINPROTEOMICSSTUDYOFPROTEININTERACTIONTHEPRINCIPLEISTHATWHENTHETARGETPROTEINANDTHEBAITPROTEINARESPECIFICALLYCOMBINED,THEBAITPROTEINBINDSTOTHETARGETPROTEINREPORTERGENEPROMOTERISUSEDTOINITIATETHEEXPRESSIONOFREPORTERGENEINYEASTCELLSIFTHEPRODUCTOFREPORTERGENEISDETECTED,THEINTERACTIONBETWEENTHETWOGENESISINDICATEDTHEREISNOINTERACTIONBETWEENTHETWOTHISTECHNIQUEWILLBEUSEDFORLARGESCALEPROTEININTERACTIONSTUDIESAFTERMICROANDARRAYINACTUALWORK,PEOPLEHAVEDEVELOPEDASINGLECOMPLEXACCORDINGTOTHEIRNEEDSCROSSINGSYSTEM,THREEHYBRIDSYSTEMANDREVERSEHYBRIDIZATIONSYSTEM,ETCANGERMAYRETALDESIGNEDASOSPROTEINMEDIATEDTWOHYBRIDSYSTEMTHEFUNCTIONOFMEMBRANEPROTEINSCANBESTUDIED,ENRICHEDWITHYEASTDOUBLEFUNCTIONSOFHYBRIDSYSTEMINADDITION,THEROLEOFTHEYEASTTWOHYBRIDSYSTEMHASALSOBEENEXTENDEDTOTHEIDENTIFICATIONOFPROTEINSTWO,PHAGEDISPLAYTECHNOLOGYADNASEQUENCEOFAMONOCLONALANTIBODYISENCODEDONABACTERIOPHAGECOATPROTEINGENE,ANDACORRESPONDINGMONOCLONALANTIBODYISEXPRESSEDONTHESURFACEWHENTHEPHAGEISGROWN,ANDTHENTHEPHAGEISPASSEDONTHECOLUMNTHETARGETPROTEINSARESPECIFICALLYBOUNDTOTHEANTIBODIES,KNOWNASPHAGEDISPLAYTECHNOLOGYTHISTECHNIQUEISALSOUSEDPRIMARILYTOSTUDYPROTEININTERACTIONS,NOTONLYHIGHTHROUGHPUTBUTALSOSIMPLETHEINVENTIONALSOHASTHEADVANTAGESOFDIRECTLYOBTAININGGENESANDHIGHLYSELECTIVESCREENINGOFCOMPLEXCOMPOUNDS,ANDINTHEPROCESSOFSCREENING,THESPECIFICITYOFTHECOMBINATIONCANBEDIRECTLYEVALUATEDBYAPPROPRIATELYCHANGINGCONDITIONSATPRESENT,USINGOPTIMIZEDPHAGEDISPLAYTECHNOLOGY,CDNALIBRARIESOFTWOSPECIFICCELLLINESOFHUMANANDMOUSEHAVEBEENDEMONSTRATEDANDISOLATEDFROMTHESIGNALPATHWAYOFHUMANEPITHELIALGROWTHFACTORSIGNALINGPATHWAYNUMBERMOLECULETHREE、PLASMARESONANCETECHNOLOGYSURFACEPLASMONRESONANCESURFACE,PLASMON,RESONANCE,SPRHASBECOMEANEWMETHODINPROTEINPROTEININTERACTIONRESEARCHITISBASEDONTHEUSEOFANANOSCALEMEMBRANEATTACHEDTOTHE“BAITPROTEIN“,WHENTHETESTPROTEINANDBAITPROTEINBINDING,THERESONANCEPROPERTIESOFTHEFILMWILLCHANGE,YOUCANSEETHROUGHTHEDETECTIONOFTHECOMBINATIONOFTHESETWOPROTEINSADVANTAGESOFSPRTECHNOLOGYITDOESNOTNEEDMARKERSORDYES,ANDTHEREACTIONPROCESSCANBEMONITOREDINREALTIMETHEASSAYISFASTANDSAFEANDCANBEUSEDTODETECTINTERACTIONSBETWEENPROTEINS,NUCLEICACIDS,ANDOTHERBIOLOGICALMACROMOLECULESFOUR,FLUORESCENCEENERGYTRANSFERTECHNOLOGYFLUORESCENCERESONANCEENERGYTRANSFERFRETISWIDELYUSEDTOSTUDYTHEDISTANCEBETWEENMOLECULESANDTHEIRINTERACTIONINCOMBINATIONWITHFLUORESCENCEMICROSCOPY,PROTEINSANDLIPIDSINLIVINGORGANISMSCANBEQUANTITATIVELYOBTAINED,TEMPORALANDSPATIALINFORMATIONOFDNAANDRNAWITHTHEDEVELOPMENTOFGREENFLUORESCENTPROTEINGFP,ITISPOSSIBLEFORFRETFLUORESCENCEMICROSCOPYTOMEASURETHEDYNAMICPROPERTIESOFLIVINGCELLMOLECULESINREALTIMEPUTFORWARDAASIMPLEMETHODFORQUANTITATIVEMEASUREMENTOFFRETEFFICIENCYANDTHEDISTANCEBETWEENDONORANDACCEPTORREQUIRESONLYASETOFFILTERSANDARATIOMEASUREMENT,ANDTHESPECTRAOFDONORANDACCEPTORAREUSEDTOELIMINATETHESPECTRACROSSTALKTHEMETHODISSIMPLEANDRAPID,ANDCANBEUSEDTOMEASURETHEEFFICIENCYOFFRETANDTHEDISTANCEBETWEENDONORANDRECIPIENTQUANTITATIVELYINREALTIME,ESPECIALLYFORDONORRECEPTORPAIRSBASEDONGFPFIVEANTIBODYANDPROTEINARRAYTECHNOLOGYTHEADVENTOFPROTEINCHIPTECHNOLOGYBRINGSNEWIDEASTOPROTEOMICSRESEARCHAMAJORCOMPONENTOFPROTEOMICSISTHESTUDYOFPROTEINLEVELSUNDERDIFFERENTPHYSIOLOGICALCONDITIONS,MINIATURIZED,INTEGRATED,HIGHTHROUGHPUTANTIBODYCHIPISAVERYGOODRESEARCHTOOL,HEISALSOTHEFASTESTGROWINGCHIPINTHECHIP,BUTALSOINTECHNOLOGYHASBECOMEINCREASINGLYMATURETHESEONESHERESOMEOFTHEANTIBODYCHIPSHAVEBEENDEVELOPEDFORCLINICALAPPLICATIONS,SUCHASTUMORMARKERS,ANTIBODYMICR