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Bpsbioscience/Anti–5–hmC polyclonal antibody (rabbit)/25205/100 µl

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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–hydroxymethylcytosine(5–hmC)conjugatedtoKLHAssayConditions:Determinationofthe5-hmCrabbitpolyclonalantibodytiterTodeterminethetiter,anELISAwasperformedusingaserialdilutionoftherabbitpolyclonalantibodydirectedagainst5-hmCinantigencoatedwells.TheantigenusedwasBSAcoupledtothe5-hmCbase.Byplottingtheabsorbanceagainsttheantibodydilution,thetiteroftheantibodywasestimatedtobe1:3,500.AnhydroxymethylatedDNAIP(hMeDIP)wasperformedusingtherabbitpolyclonalantibodydirectedagainst5-hydroxymethylcytosine(Cat.No.25205).TheIgGisotypeantibodiesfromrabbitwereusedasnegativecontrol.TheDNAwaspreparedwiththeGenDNAmoduleofthehMeDIPkitandsonicatedwtohaveDNAfragmentsof300-500bp.1μgofhumanHeLacellsDNAwerespikedwithnon-methylated,methylated,andhydroxymethylatedfragments.TheimmunoprecipitatedmaterialhasbeenanalysedbyqPCRusingtheprimerpairspecificforthe3differentcontrolsequences.Theobtainedresultsshowthattherabbitpolyclonalfor5-hmCishighlyspecificforthisbasemodification(noIPwithnon-methylatedormethylatedCbasescontainingfragments).Dotblotanalysisofthe5-hmCrabbitpolyclonalantibodywiththeC,mCandhmCPCRcontrols100to4ng(equivalentof5to0.2pmolofC-bases)ofthehmC,mCandCPCRcontrolswerespottedonamembrane(AmershamHybond-N+).Themembranewasincubatedwiththerabbit5-hydroxymethylcytosinepolyclonalantibody(dilution1:200).Themembraneswereexposedfor30seconds.Concentration:100µlFormulation:Wholeantiserumfromrabbitcontaining0.05%azideSpeciesReactivity:Human,mouse,broadrangePurification:WholeserumImmunogen:nucleotideFormat:aqueoussolutionStorage/StABIlity:Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.Application(s):hMeDIP(2.5µg/IP)ELISA(1:500)DB(1:200)Notes:Theoptimalantibodyconcentrationshouldbedeterminedbytheend–user.Warning(s):Avoidfreeze/thawcyclesScientificCategory:AntibodiesProductType:AntibodyDatashownislot-specific.Contactusforspecificinformationonotherlots.

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