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Bpsbioscience/Anti–5–hmC polyclonal antibody (rabbit)/25203/50 µg

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25203

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HostSpecies:RabbitClonality:polyclonalBackground:5–hydroxymethylcytosine(5–hmC)resultsfromtheenzymaticconversionof5–methylcytosineinto5–hydroxymethylcytosinebytheTETfamilyofiron–dependentoxygenases.5–hmCbaseswererecentlydiscoveredinmammalianDNA,inPurkinjeneurons,ingranulecellsandembryonicstemcellswheretheyarepresentathighlevels(upto0.6%oftotalnucleotidesinPurkinjecells).Recentreportsindicatethat5–hmCisabundantinbraintissue,especiallyinareasthatareassociatedwithhighercognitivefunctions.Preliminaryresultsindicatethat5–hmCmayhaveimportantrolesdistinctfrom5–mC.Althoughitspreciserolehasstilltobeshown,earlyevidencesuggests5–hydroxymethylcytosinemayrepresentanewanduniquepathwaytodemethylateDNAinvolvingarepairmechanismconverting5–hmCtocytosine.Duetothestructuralsimilaritybetween5–mCand5–hmC,thesebasesareexperimentallyalmostindistinguishable.Themostcommonlyusedmethodologies(e.g.enzymaticapproaches,bisulfitesequencing)donotdistinguish5mcfrom5–hmC.Thedevelopmentofspecificantibodiesappearstobethemostpowerfulwaytodistinguishandspecificallyenrichfor5–mCand5–hmCsequences.Description:Polyclonalantibodyraisedinrabbitagainst5–hydroxymethylcytosineconjugatedtoKLH.AssayConditions:hMeDIPresultsobtainedwiththeantibodydirectedagainst5-hmChMeDIP(hydroxymethylatedDNAIP)wasperformedusingtheantibodyagainst5-hydroxymethylcytosine(Cat.No.25203).DNAfrommouseEScellswaspreparedwiththeGenDNAmoduleofthehMeDIPkitandsonicatedtoobtainDNAfragmentsof300-500bp.OneμgofshearedDNAwasspikedwithunmethylated(C)methylated(mC),andhydroxymethylated(hmC)controls.hMeDIPwasperformedwith3.5μgoftherabbit5-hmCantibodyandtheimmunoprecipitatedDNAwasanalysedbyqPCRusingprimersspecificforthe3differentcontrolsequences.Figure1showsthattherabbitpolyclonalantibodyagainst5-hmCishighlyspecificforthe5-hmCbasemodification(noIPwithnon-methylatedormethylatedCcontrolfragments).DeterminationoftheantibodytiterTodeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainst5-hmC(cat.No.25203),crudeserumandflowthrough,inantigencoatedwells.Theantigenusedwasthe5-hmCbasecoupledtoBSA.Byplottingtheabsorbanceagainsttheantibodydilution,thetiteroftheantibodywasestimatedtobe1:2,800.Dotblotanalysisusingtheantibodydirectedagainst5-hmCTodemonstratethespecificityoftheantibodyagainst5-hmC(cat.No.25203),aDotblotanalysiswasperformedusinghmC,mCandCcontrols.Onehundredto4ng(equivalentof5to0.2pmolofC-bases)ofthecontrolswerespottedonamembrane(AmershamHybond-N+).Theantibodywasusedatadilutionof1:1,000.Figure3showsahighspecificityoftheantibodyforthehydroxymethylatedcontrol.Concentration:50µgFormulation:PBScontaining0.05%azideand0.05%ProClin300.SpeciesReactivity:Human,mouse,broadrangePurification:AffinitypurifiedImmunogen:nucleotideFormat:AqueousbuffersolutionStorage/StABIlity:Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.Application(s):hMeDIP(3.5µg/IP)ELISA(1:100–1:500)DB(1:1000)Notes:Theoptimalantibodyconcentrationshouldbedeterminedbytheend–user.Warning(s):Avoidfreeze/thawcyclesScientificCategory:AntibodiesProductType:AntibodyDatashownislot-specific.Contactusforspecificinformationonotherlots.

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