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Bpsbioscience/Anti–5–caC polyclonal antibody/25201/20 µg

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25201

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HostSpecies:RabbitClonality:polyclonalBackground:Theso-called6thbase,5–hydroxymethylcytosinehasbeendiscoveredin2009.Thefunctionofthisnewmodifiedbaseremainsmostlyunclear,butitiscertainthatthe5–hmCbaseisgeneratedbyenzymaticconversionof5–methylcytosine(5–mC)into5–hydroxymethylcytosinebytheTETfamilyofoxygenases.Earlyreportssuggestedthat5–hmCmayrepresentanintermediateofactivedemethylationinanewpathwaywhichdemethylatesDNA,converting5–mCtocytosine.Recentevidencefuelthehypothesisthat5–hmCrepresentsanintermediate,whichcouldinvolvefurtheroxidationofthehydroxymethylgrouptoaformylorcarboxylgroupfollowedbyeitherdeformylationordecarboxylation.Thecarboxylandformylgroupsof5–formylcytosine(5–fC)and5–carboxylcytosine(5–caC)couldbeenzymaticallyremovedwithoutexcisionofthebase.Description:Polyclonalantibodyraisedinrabbitagainst5–Carboxylcytosine(5ca–CMPmonophosphate)conjugatedtoBSASynonym(s):5–CarboxylcytosineAssayConditions:Dotblotanalysisusingtheantibodydirectedagainst5-caCTodemonstratethespecificityoftheantibodyagainst5-caC(Cat.#25201),aDotBlotanalysiswasperformedusingsyntheticoligonucleotidescontainingdifferentmodifiedC-bases(indicatedinred).125and25ngoftherespectiveoligoswereboundtoaStreptavindin-coatedmulti-wellplate.Theantibodywasusedatadilutionof1:1,000.ThebindingofantibodytotheDNAwasmeasuredbyECLchemiluminescence.Figure1showsahighspecificityoftheantibodyforthecarboxylatedcytosine.Immunofluorescenceassayusingtheantibodydirectedagainst5-caC293TcellsweretransfectedwitheitherthemouseFLAG-taggedwild-typeTet1(Tet1CD)orthecatalyticallyinactiveFLAG-taggedC-terminaldomainofTet1(Tet1mCD)andstainedwiththeantibodyagainst5-caC(Cat.#25201),diluted1:500,andwithananti-FLAGantibody,followedbyDAPIcounterstaining.Immunoprecipitationusingtheantibodydirectedagainst5-caCImmunoprecipitationwasperformedwiththeantibodyagainst5-caC(Cat.#25201)on2µgofJ1ESgenomicDNA,spikedwith1pgofacontrolDNAfragment(approximately700bpfromtheRFP(Ringfingerprotein)gene)containingdifferentcytosinemodifications.ThemCandhmCcontrolDNAwasgeneratedbyPCRwiththecorrespondingnucleotide.ThecaCcontrolfragmentwasobtainedbyinvitromethylationusingM.SssImethyltransferasefollowedbyoxidationwithpurifiedTet2.TheimmunoprecipitatedDNAwassubsequentlyanaysedbyqPCRusingprimersspecificforthecontrolDNAfragmentsandforGAPDH,usedasanegativecontrol.Figure3showstheenrichmentcalculatedastheratiooftherecoveryofthecontrolDNAversustherecoveryoftheGAPDHnegativecontrol. Concentration:20µgFormulation:PBS(pH7.4),30%glycerol,0.05%sodiumazideSpeciesReactivity:NACrossReactivity:Specificfor5–carboxylcytosine(5–caC).Doesnotreactwith5–mc,5–hmc,unmodifiedcytosine,or5–formylcytosine(5–fC)and5–carboxylcytosine(5–caC)Purification:ProteinAaffinitypurifiedImmunogen:nucleotideFormat:AqueousbuffersolutionStorage/StABIlity:Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.Application(s):DB(1:500–1:1000)IF(1:500)DNAIP(4µggenomicDNA/IP)Notes:Theoptimalantibodyconcentrationshouldbedeterminedbytheend–user.Warning(s):Avoidfreeze/thawcyclesScientificCategory:AntibodiesProductType:AntibodyDatashownislot-specific.Contactusforspecificinformationonotherlots.

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