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®ENDEXT®TechnologyWEPRO7240/7240H/7240GExpressionKitInstructionmanualforproteinsynthesiswithwheatgermcell-freesystem(CatalogNo.CFS-TRI-7240,CFS-TRI-7240H,CFS-TRI-7240G)CellFreeSciencesCo.,Ltd.1®1.IntroductionWEPRO7240ExpressionKitWEPRO7240ExpressionKitcontainsWEPRO®7240.Itissuitableforproteinexpressionwhereaffinitypurificationtohighpuritiesisneithernecessarynordesirable.WEPRO7240HExpressionKitWEPRO7240HExpressionKitissuitableforHis-taggedproteinexpressionandpurification.Thepost-purificationpurityoftheproteinexpressedwiththiskitishigherthanthatwithWEPRO7240ExpressionKit.PleasenotethattheyieldofproteinwiththiskitisslightlylessthanthatwithWEPRO7240ExpressionKit.ForthepurificationofHis-taggedproteinexpressedusingthiskit,Ni-sepharosehighperformanceresinofGEHealthcareisrecommended.Pleasenotethat1xSUB-AMIX®SGCandWEPRO®7240Hinclude4mMdithiothreitol(DTT)andthatsomeotherresinsarenotresistanttoDTTofthisconcentration.Forexample,Ni-NTAofQiagenisnotrecommendedforthisreason.Pleaseensurethatyourresinisresistantto4mMDTT.WEPRO7240GExpressionKitWEPRO7240GExpressionKitissuitableforGST-taggedproteinexpressionandpurification.Thepost-purificationpurityoftheproteinexpressedwiththiskitishigherthanthatwithWEPRO7240ExpressionKit.PleasenotethattheyieldofproteinwiththiskitisslightlylessthanthatwithWEPRO7240ExpressionKit.ForthepurificationofGST-taggedproteinexpressedusingthiskit,glutathionesepharose4BresinofGEHealthcareisrecommended.Pleasenotethat1xSUB-AMIX®SGCandWEPRO®7240Ginclude4mMdithiothreitol(DTT)andthatsomeotherresinsarenotresistanttoDTTofthisconcentration.Pleaseensurethatyourresinisresistantto4mMDTT.2®2ProtocolOverviewTranscriptionofplasmidDNAIncubationat37°Cfor6hours(seeSection4.3.1or4.4.1or4.5.1)AdditionalpurificationofplasmidDNA(optional)(seeSection4.2)DetectionoftargetproteinbySDS-PAGE(seeSection4.6)TranslationoftargetproteinbybilayerreactionIncubationat15°Cfor20hours(seeSection4.3.2or4.4.2or4.5.2)PlasmidDNAconstruction(seeSection4.1)3®3Materials3.1ContentsoftheKit3.2InstructionfortheUseofReagentsItemDescriptionStorageWEPRO®7240/WEPRO®7240H/WEPRO®7240GWEPRO®7240/7240H/7240G(wheatgermextract)issensitivetotemperatureandvibration.Immediatelyafterthawingunderrunningwater,placethereagentonice.Uponthawingforthefirsttime,separatetheportionthatisnotusedimmediately,andtoavoidmultiplefreeze-thawingexposures,subdivideitintoappropriatevolumesinseparatecontainers.Storethemat-80°Cforlateruse.Donotsubjectitto3ormorefreeze-thawingcycles.Afterthethirdfreeze-thawingcycle,itispossiblethatproteinsynthesisactivitydecreases,thedegreeofwhichdependsonthewayofhandling.Useofliquidnitrogenisrecommendedforre-freezing.Whenusingthereagent,mixitgentlybypipettingseveraltimes.Avoidbubbling.-80°CSUB-AMIX®SGC(S-1,S-2,S-3,S-4)Thisproductconsistsofasetof4reagents(S-1,S-2,S-3,S-4)at40xconcentration.Storeall4reagentsat-20°Corbelow.Nochangeintheirreactionefficiencyhasbeenobservedafter10freeze-thawingcycles.Toprepare44mlof1xSUB-AMIX®SGCmixture,add1.1mleachofS-1throughS-4to39.6mlofnuclease-freewaterwhileagitatingthelatter.If4reagentsaremixedfirst,precipitationmayoccur.Onceithappens,ittakestimetodissolvetheprecipitates.Toavoidmultiplefreeze-thawingexposures,subdivide1xSUB-AMIX®SGCmixtureintoappropriatevolumesinseparatecontainersandstorethemat-80°C.Donotsubject1xSUB-AMIX®SGCmixturetomultiplefreeze-thawingcycles.Decreaseinthereactionefficiencymayoccur,thedegreeofwhichdependsonthewayofhandling.-20°Cor-80°C(Continued)ItemQuantityConcentrationVolumeWEPRO®7240/WEPRO®7240H/WEPRO®7240G2240OD/ml1mlSUB-AMIX®SGCS-1140x1.1mlSUB-AMIX®SGCS-2140x1.1mlSUB-AMIX®SGCS-3140x1.1mlSUB-AMIX®SGCS-4140x1.1ml5xTranscriptionBufferLM15x0.4mlNTPMix125mM0.2mlSP6RNAPolymerase180U/µl30µlRNaseInhibitor180U/µl30µlCreatineKinase120mg/ml20µlpEU-E01-MCS11µg/µl10µlpEU-E01-DHFR11µg/µl10µl4®(Continuedfromsection3.2)ItemDescriptionStorage5xTranscriptionBufferLMAfterthawing,subdivide5xTranscriptionBufferLMintoappropriatevolumesconvenientforyouruse.Ithasbeenconfirmedthatthefreeze-thawingcycleforthisproductcanberepeatedupto10times.-20°CNTPMixATP,GTP,CTP,andUTPinthisNTPMixhaveallbeenpreparedataconcentrationof25mM.Afterthawing,subdividetheNTPMixintoappropriatevolumesconvenientforyouruse.Ithasbeenconfirmedthatthefreeze-thawingcycleforthisproductcanberepeatedupto10times.-20°CSP6RNAPolymerase50%glycerolisincluded.-20°CRNaseInhibitor50%glycerolisincluded.-20°CCreatineKinase(*1)CreatineKinaseisrequiredforproteinexpression.Avoidmultiplefreeze-thawingexposures;otherwise,theactivityofCreatineKinasemaydecrease.TheconcentrationofthestartingCreatineKinasesolutionis20mg/ml.Useitasitisforpreparingthetranslationmixtureforlargescaleandmiddlescaletranslation.Forsmallscaletranslation(seeSection4.3.2),diluteitwithnuclease-freewaterto1mg/ml.Inbothcases,thefinalconcentrationofCreatineKinaseintranslationmixtureshouldbe40ng/µl.-80°CpEU-E01-MCSExpressionvectorforsubcloningyourgeneofinterest.Seesection4.1indetail.-20°CpEU-E01-DHFRExpressionvectorencodingdihydrofolatereductase(DHFR)genederivedfromE.coli.Itworksaspositivecontrolforprotei