HostSpecies:MouseClonality:clone33D3Isotype:IgG1Background:5–Methylcytosine(5–Methylcytidine)isamodifiedbasethatisfoundintheDNAofplantsandvertebrates.DNAmethylationisanepigeneticeventinwhichDNAmethyltransferases(DNMTs)catalyzethereactionofamethylgrouptothefifthcarbonofcytosineinaCpGdinucleotide.Thismodificationhelpstocontrolgeneexpressionandisalsoinvolvedingenomicimprinting,whileaberrantDNAmethylationisoftenassociatedwithdisease.The5–methylcytidineantibody(Clone33D3)hasbeendevelopedtodiscriminatebetweenthemodifiedbaseanditsnormalcytosinecounterpart,allowingforgenepromotermethylationanalysis.Description:Monoclonalantibodyraisedinmouseagainst5–mC(5–methylcytosine)conjugatedtoovalbumin.The5–methylcytosineantibody(clone33D3)isthemostpublishedandwidelyusedantibodyforDNAmethylationanalysis.IthasbeenvalidatedforMethylatedDNAImmunoprecipitation(MeDIP–seq,MeDIP–on–chip),Immunofluorescence,FlowCytometry,Dotblot&ELISA.AssayConditions:MeDIP-seq(methylatedDNAimmunoprecipitation-sequencing)withthemonoclonalantibodydirectedagainst5-mCGenomicDNAfromE14EScellswasshearedtogeneraterandomfragments(sizerange300to700bp).OneμgofthefragmentedDNAwasligatedtoIlluminaadaptersandtheresultingDNAwasusedforastandardMeDIPassay,using2μgofthemonoclonalagainst5-mC(Cat.#25206).AfterrecoveryofthemethylatedDNA,IlluminasequencinglibrariesweregeneratedandsequencedonanIlluminaGenomeAnalyzeraccordingtothemanufacturer’sinstructions.Figure1Aand1BshowGenomebrowserviewsofCAsimplerepeatelementswithreaddistributionsspecificfor5-mCat2genelocations(SigleC15andMfsd4).VisualinspectionofthepeakprofilesinagenomebrowserrevealshighenrichmentofCAsimplerepeatsinaffinity-enrichedmethylatedfragmentsafterMeDIPwiththe5-mCmonoclonalantibody.MeDIPresultsobtainedwiththemonoclonalantibodydirectedagainst5-mCMeDIP(MethylatedDNAimmunoprecipitation)wasperformedonfragmentedhumangenomicDNAusingthemonoclonalantibodyagainst5-mC(Cat.#25206).ThefragmentedDNAwasspikedwiththeinternalcontrolspresentinthekit(methylatedDNA(meDNA)asapositiveandunmethylatedDNA(unDNA)asanegativecontrol)priortoperformingtheIP.QPCRwasperformedwithoptimizedprimersets,includedinthekit,specificforthemethylatedandunmethylatedDNAcontrols,andforaknownmethylated(TSH2B)andunmethylated(GAPDH)genomicregion.Anadditionalinternalpositiveandnegativecontrollocus(4994+and8804-,respectively)werealsotested(4994+:forwardprimer5’-GGGAATATAAGGAGCGCACA-3’andreverseprimer5’-TCGGTTAAAACGGTCAGGTC-3’;8804-:forwardprimer5’-CGAGGCGTGAGTTATTCCTG-3’andreverseprimer5’-CTCTTGTGGCTGAGCTCCTT-3’).Figure2showstherecovery(meanof3experiments),expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).ELISAusingthemonoclonalantibodydirectedagainst5-mCELISAwasperformedusingmonoclonalantibodyagainst5-mC(Cat.#25206),diluted1:100.Thewellswerecoatedwithaserialdilutionofhydroxymethylated(hmC),methylated(mC)andunmethylated(C)DNAstandards.Dotblotanalysisusingthemonoclonalantibodydirectedagainst5-mCTodemonstratethespecificityoftheantibodyagainst5-mC(Cat.#25206),aDotblotanalysiswasperformedusinghydroxymethylated(hmC),methylated(mC)andunmethylated(C)DNAstandards.Onehundredto4ng(equivalentof5to0.2pmolofC-bases)ofthecontrolswerespottedonamembrane.Theantibodywasusedatadilutionof1:250.Figure3showsahighspecificityoftheantibodyforthemethylatedcontrol.Immunofluorescenceresultsobtainedwiththemonoclonalantibodydirectedagainst5-mCFigure5A:HumanHeLacellswerestainedwiththemonoclonalantibodyagainst5-mC(Cat.#25206).Cellswerefixedwith4%formaldehydeinPBSfor10min.atroomtemperature,permeABIlisedwith0.5%TritonX-100for1hourandtreatedwith2NHClfor1hour.AfterblockingwithPBScontaining0.1%TritonX-100and1%BSA,thecellswereimmunofluorescentlylabeledwiththe5-mCantibody(left)diluted1:500inblockingsolution,followedbyagoatanti-mouseantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright.Figure5B:ImmunofluorescentstainingofaninterphaseHeLacellwiththe5-mCantibodyfollowedbyagoatanti-mouseantibodyconjugatedtoFITC(yellow)andwithHoechststaining(blue).Surfaceplasmonresonance(SPR)analysisofthethemonoclonalantibodydirectedagainst5-mCAsynthesizedbiotin-labeled5-mCconjugatewasimmobilizedonaCM4BIAcoresensorchip(GEHealthcare,France).Briefly,twoflowcellswerepreparedbysequentialinjectionsofEDC/NHS,streptavidin,andethanolamine.Oneoftheseflowcellsservedasnegativecontrol(biotinylatedspacerwithout5-mC),whilebiotinylated5-mCconjugatewasinjectedintheotherone,toobtainanimmobilizationlevelof55responseunits(RU).AllSPRexperimentswereperformedusingHBS-Nbuffer(10mMHEPES,150mMNaCl,pH7.4)ataflowrateof5μl/min.Interactionassaysinvolvedinjectionsof3differentdilutionsofthe5-mCmonoclonalantibody(Cat.#25206)overthebiotinylated5-mCconjugateandnegativecontrolsurfaces,followedbya3minwashingstepwithHBS-Nbuffertoallowdissociationofthecomplexesformed.Attheendofeachcycle,thestreptavidinsurfacewasregeneratedbyinjectionof0.1Mcitricacid,pH3.Thesensorgramscorrespondtothebiotinylated5-mCconjugatesurfacesignalsubtractedwiththenegativecontrol.DatafromthesensorgramsthatreachedbindingequilibriumwereusedforScatchardanalysis.Thevalueofthedissociationconstant(kd)obtainedbyglobalfittingand1:1Langmuirmodelis13±9nM.Concentration:10µg/5µlFormulation:PBScontaining0.05%azideSpeciesReactivity:Human,mouse,broadrangeCrossReactivity:Reactswith5–mCfoundinallvertebrateandplantspecies.Doesnotcross–reactwithothermodifiedcytosines.Purification:PurifiedbygelfiltrationImmunogen:nucleotideFormat:AqueousbuffersolutionStorage/Stability:Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.Application(s):MeDIP(1–2µg/IP)ELISA(1:100)DB(1:250)IF(1:500)FISH(1:500)Southernblot(1:200)Notes:Theoptimalantibodyconcentrationshouldbedeterminedbytheend–user.Warning(s):Avoidfreeze/thawcyclesScientificCategory:AntibodiesProductType:AntibodyDatashownislot-specific.Contactusforspecificinformationonotherlots.