DescriptionTheamyloidbetapeptideisderivedfromthecleavageoftheAmyloidprecursorprotein(APP)andvariesinlengthfrom39to43aminoacids.However,theform(s)ofamyloid-betapeptide(Aβ)associatedwiththepathologycharacteristicofAlzheimer’sdisease(AD)remainsunclear.Inparticular,theneurotoxicityofintraneuronalAβaccumulationisanareaofconsiderableresearchandcontroversyprincipallybecauseantibodiesthoughttobespecificforAβhavebeenshowntoactuallydetectintraneuronalAPPandnotAβexclusively.MOAB-2(mouseIgG2b)isapan-specific,high-titerantibodytoAβresidues1-4asdemonstratedbybiochemicalandimmunohistochemicalanalyses(IHC),andishighlyspecificjusttoamyloidbetapeptide.MOAB-2didnotdetectAPPorAPP-CTFsincellculturemedia/lysates(HEK-APPSweorHEKAPPSwe/BACE1)orinbrainhomogenatesfromtransgenicmiceexpressing5familialAD(FAD)mutation(5xFADmice).UsingIHCon5xFADbraintissue,MOAB-2immunoreactivityco-localizedwithC-terminalantibodiesspecificforAβ40andAβ42.MOAB-2didnotco-localizewitheitherN-orC-terminalantibodiestoAPP.Inaddition,noMOAB-2-immunreactivitywasobservedinthebrainsof5xFAD/BACE-/-mice,althoughsignificantamountsofAPPweredetectedbyN-andC-terminalantibodiestoAPP,aswellasby6E10.Inboth5xFADand3xTgmousebraintissue,MOAB-2co-localizedwithcathepsin-D,aMarkerforacidicorganelles,furtherevidenceforintraneuronalAβ,distinctfromAβassociatedwiththecellmembrane.MOAB-2demonstratedstrongintraneuronalandextra-cellularimmunoreactivityin5xFADand3xTgmousebraintissues.BiosensisnowoffersbiotinylatedMOAB-2antibodyallowingmoreflexibilityinexperimentaldesignbyusingthebiotin-avidin/streptavidindetectionmethod.BiotinylatedMOAB-2antibodymayalsohelptoreducebackgroundstainingindifficult-to-staintissuesandincreasedetectionsensitivity.TheABIlityofbiotinylatedMOAB-2antibodytodetectamyloidbetahasbeenvalidatedbyIHC.Purified,non-biotinylatedMOAB-2antibodyisavailablehere.RelatedproductsPurified,non-biotinylatedMOAB-2antibody,cat#M-1586-100OligomericAβELISAKit,cat#BEK-2215-1P/2PBatchNo.SeeproductlabelUnitsize50µgAntigenRecombinanthumanamyloidbetaprotein42(Aβ42):DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAAntigenLocationEpitopemapstoresidues1-4ofhumanamyloidbetapeptide40/42AntigenLength42aminoacidsAntibodyTypeMousemonoclonalIsotypeIgG2b,lamBDaCloneMOAB-2OtherNamesBeta-APP42;Beta-APP40;Beta-amyloidprotein42;Beta-amyloidprotein40;ABPP;APPI;AmyloidbetaA4protein;MOAB2;MOAB-2;Alzheimer"santibody;AB40;AB42;abetaAccessionP05067A4_HUMANProducedinMouseMolecularWeightWithFormicacidextractionsandstandardreducingwesternblottingprocedures,beta-amyloidpeptidemigratesbetween3-6kDa(seeFigure1).PurityAntibodywaspurifiedfromcellculturesupernatantbyProteinGchromatography,biotinylatedandbuffer-exchangedintoPBS,pH7.4bufferApplicationsThebiotinylatedMOAB-2antibodyhasbeentestedbyIHC(1:500–1:2,000dilution)andisalsoexpectedtoworkinapplicationsvalidatedfortheunlabelledantibody(M-1586-100)atsameorhigherdilutions:WesternBlotting(WB),Immunohistochemistry(IHC),Immunohistochemistry/paraffinembeddedIHC(P),Immunoprecipitation(IP),Immunofluorescence(IF),ELISA.WesternBlotting:MOAB-2hasbeentestedinWBusingpurifiedsyntheticbeta-amyloidpreparationsandfromtransgenicmousebrainformicacidextracts(seeFigure1).Formicacidextraction/concentrationisrequiredforwesternblotdetectionfromextracts.Suggesteddilutionof1:2000-1:5,000forWB,standardECLdetectionsystems.Tissuesamplesforthedetectionofbeta-amyloidshouldbepreparedasdetailedinYoumansKLetal.,2011(JournalofNeuroscienceMethods196:51–59)forbestresults.Detectionofbeta-amyloid40/42indirectwesternscanbedifficult;Dot-blotsofpreparedsamplesarerecommendedasdetailedinYoumansKLetal.,2012.Immunohistochemistry:SuggesteddilutionforbiotinylatedMOAB-2inIHCis1:500-1:2,000.Freshfrozen,4%paraformaldehydefixedfrozen,orformalinfixedparaffinembeddedtissuesareallsuitable.Antigenretrievalisrequiredinfixedtissuesforoptimalstaining.Antibodywastestedon4%paraformaldehyde/0.1%glutaraldehydefixedfrozentissuefrom3xTgand5xFADmice.MOAB-2antibodydetectsintraneuronalandextracellularbeta-amyloidinIHCanddoesnotdetectAPP(YoumansKLetal.,2012).Theantibodyalsoreactswitharchivalformalin-fixed,paraffin-embeddedtissuesampleswithantigenHeatInducedEpitopeRetrieval(HIER).RecommendedbufferforHIERiscitrate,pH6.0.Signalwasweakwithoutantigenretrieval.Immunoreactivitywasobservedinintraneural-amyloiddeposition(plaque)inAlzheimer’sbrain.MOAB-2wasfoundtobeextremelycleanandwithanexcellentsignaltonoiseratiowithnoneuro-cellulardiffusivestaining.Inaddition,MOAB-2demonstratednosignificantdifferencesinAβdetectionusingparaffinfixed,free-floatingsections(YoumansKLetal.,2012).Formicacid(FA)treatmentresultedinoptimaldetectionofbothintraneuronalandextracellularAβcomparedtowithoutFA(incubatedin88%FA8min,YoumansKLetal.,2012).FreefloatingtissuesectionswerepermeabilizedinTBScontaining0.25%TritonX-100(TBSX;3×10min),blockedwith3%horseseruminTBSX(3×10min)followedby1%horseseruminTBSX(2×10min)andincubatedwithappropriateprimaryantibodiesdilutedinTBSXcontaining1%horseserumovernight.SeeYoumansKLetal.,2012,forfullIHC(P)protocolandmethoddetails.Immunofluorescence:ForIF,suggesteddilutionis1:100-1:500.Theantibodywastestedon4%PFAfixedfrozentissue.FixedtissueswerewashedinTBS(3×10min),thenincubatedin88%FA(8min),andthenpermeabilizedinTBSX(3×10min),andblockedinTBSXcontaining5%bovineserumalbumin(BSA;1hr).SectionsweresubsequentlyincubatedwithappropriateprimaryantibodiesdilutedinTBSXcontaining2%BSAovernightonanoscillatoryrotator.Detectionwasviafluorescentlylabelledabsorbedsecondaryantibodies(YoumansKLetal.,2012).Immunoprecipitation:ForIP,thesuggesteddilutionis1:200to1:1,000forlabelledbeta-amyloidusingSA-coatedbeadsasthecapturevehicle,similartotheprotocolsemployedbyYoumansKLetal.,2012.ELISA:InanELISA,adilutionof1:50-1:1,000issuggested.TheantibodyhasbeentestedinELISAsonsyntheticbeta-amyloidandtissuehomogenatesfrombeta-amyloid-Tgmice.Biosensisrecommendsoptimaldilutions/concentrationsshouldbedeterminedbytheenduserforallapplications.Dilutionsprovidedareonlymeanttoserveasabasicguide.SpecificityMOAB-2detectspreparationsenrichedinU-,O-,F-Aβ42,andU-Aβ40bydot-blot,andisthusapan-specificAβantibody.However,MOAB-2isselectiveforthemoreneurotoxicAβ42comparedtoAβ40.Indeed,MOAB-2demonstratedatitrationagainstantigenconcentration,anddetectsAβ40at2.5pmol,butU-,O-andF-Aβb42atantigenconcentrationsaslowas~0.1pmol(Youmans.KLetal.,2012;PMID:22423893).MOAB-2doesnotdetectAPP(AmyloidPrecursorProtein).SpeciesAgainstHumanAntibodyAgainstHumanCross-reactivityHuman,rat,otherspeciesnotyettested.ByDotBlot,MOAB-2detectedratAβ40andhumanAβ40,albeitwithlessaffinitythanforAβ42(YoumansKLetal.,2012).BlastURLClickhereConjugateBiotinFormLyophilizedfromPBSbuffer,pH7.4;containsnopreservative.AppearanceDrypowder.ReconstitutionSpinvialbrieflybeforeopening.Reconstitutein50µLofsterilewatertogiveaconcentrationof1mg/mL.Centrifugetoremoveanyinsolublematerial.FinalbufferisPBS,pH7.4withoutpreservative.StorageAfterreconstitutionkeepaliquotsat-20°Cto-70°Cforahigherstability.At2-8°Ckeepuptooneweek;usesterilemethodsandPipettes.Highlypurifiedglycerol(1:1)maybeaddedforanadditionalstability.Avoidrepetitivefreeze/thawcycles.Keeptightlyclosedwhennotinuseandprotectedfromlight.ExpiryDate12monthsafterpurchase.SpecificReferencesRuan,CS.etal.(2017)Sortilininhibitsamyloidpathologybyregulatingnon-specificdegradationofAPP.ExpNeurol.[Epubaheadofprint]Application:IHCReferencesfornon-biotinylatedMOAB-2antibody(M-1586-100):Zhu,B.etal.(2017)ER-associateddegradationregulatesAlzheimer"samyloidpathologyandmemoryfunctionbymodulatingγ-secretaseactivity.NatCommun.8(1):1472.Application:IHCHuang,TY.etal.(2017)SORLAattenuatesEphA4signalingandamyloidβ–inducedneurodegeneration.JExpMed.pii:jem.20171413.[Epubaheadofprint].Application:IHCFelecia,M.etal.(2017)PeripheralInflammation,ApolipoproteinE4,andAmyloid-βInteracttoInduceCognitiveandCerebrovascularDysfunction.ASNNeuro.9(4):1759091417719201.Application:IHC/IFThomas,R.etal.(2016)EpidermalgrowthfactorpreventsAPOE4andamyloid-beta-inducedcognitiveandcerebrovasculardeficitsinfemalemice.ActaNeuropatholCommun.4(1):111Application:IHCKoster,KP.etal.(2016)Epidermalgrowthfactorpreventsoligomericamyloid-βinducedangiogenesisdeficitsinvitro.JCerebBloodFlowMetab.[Epubaheadofprint]Application:IFLöffler,T.etal.(2016)DecreasedPlasmaAβinHyperlipidemicAPPSLTransgenicMiceIsAssociatedwithBBBDysfunction.Front.Neurosci.Application:IFKobro-Flatmoen,A.etal.(2016)Reelin-immunoreactiveneuronsinentorhinalcortexlayerIIselectivelyexpressintracellularamyloidinearlyAlzheimer"sdisease.NeuroBIOLOGyofDisease.93:172-183.Application:IHCTai,LM.etal.(2016)TheroleofAPOEincerebrovasculardysfunction.ActaNeuropathol.131(5):709-23.Application:IFKim,YH.etal.(2015)A3DhumanneuralcellculturesystemformodelingAlzheimer"sdisease.NatProt.10(7):985-1006.Application:WBCondello,C.etal.(2015)MicrogliaconstituteabarrierthatpreventsneurotoxicprotofibrillarAβ42hotspotsaroundplaques.NatCommun.6:6176.Application:IFIulitaMFetal(2014)IntracellularAbetapathologyandearlycognitiveimpairmentsinatransgenicratmodeloverexpressinghumanamyloidprecursorprotein:amultidimensionalstudy.ActaNeuropatholCommun.6:61.Application:IF,IHSmithBRetal(2014)Neuronalinclusionsofalpha-synucleincontributetothepathogenesisofKrabbedisease.JPathol.Apr;235(5):509-21.Application:IFGeneralReferencesTaiLMetal(2016)"TheroleofAPOEincerebrovasculardysfunction."ActaNeuropathol.2016Feb16.[Epubaheadofprint]TaiLMetal(2013)LevelsofsolubleapolipoproteinE/amyloid-β(Aβ)complexarereducedandoligomericAβincreasedwithAPOE4andAlzheimerdiseaseinatransgenicmousemodelandhumansamples.JBiolChem.2013Feb22;288(8):5914-26.K.L.Youmansetal(2012)IntraneuronalAbetadetectionin5xFADmicebyanewAbeta-specificantibodyMolNeurodegener.2012Mar16;7(1):8.K.L.Youmansetal(2011)Amyloid-β42altersapolipoproteinEsolubilityinbrainsofmicewithfivefamilialADmutationsJNeurosciMethods.2011Mar15;196(1):51-9.