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Bpsbioscience/Anti–5–hmC monoclonal antibody (mouse)/25202/20 µg

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25202

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HostSpecies:MouseClonality:monoclonalBackground: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:Monoclonalantibodyraisedinmouseagainst5–hydroxymethylcytosineconjugatedtoBSAAssayConditions:AnhydroxymethylatedDNAIP(hMeDIP)wasperformedusingthemousemonoclonalantibodydirectedagainst5-hydroxymethylcytosine(Cat.#25202).TheIgGisotypeantibodiesfrommousewereusedasnegativecontrol.TheDNAwaspreparedwiththeGenDNAmoduleofthehMeDIPkitandsonicatedtohaveDNAfragmentsof300-500bp.1μgofhumanHeLacellsDNAwerespikedwithnon-methylated,methylated,andhydroxymethylatedPCRfragments.TheimmunoprecipitatedmaterialwasanalyzedbyqPCRusingtheprimerpairspecificforthe3differentcontrolsequences.Theobtainedresultsshowthatthemousemonoclonalfor5-hmCishighlyspecificforthisbasemodification(noIPwithnon-methylatedormethylatedCbasescontainingfragments).Determinationofthe5-hmCmousemonoclonalantibodytiterTodeterminethetiter,anELISAwasperformedusingaserialdilutionofthemousemonoclonalantibodydirectedagainst5-hmC(Cat.#25202)inantigencoatedwells.TheantigenusedwasKHLcoupledto5-hmCbase.Byplottingtheabsorbanceagainsttheantibodydilution,thetiteroftheantibodywasestimatedtobe1:40,000.Dotblotanalysisofthe5-hmCmousemonoclonalantibodywiththeC,mCandhmCPCRcontrols200to2ng(equivalentof10to0.1pmolofC-bases)ofthehmC(1),mC(2)andC(3)PCRcontrolsfrom5-hmC,5-mC&cytosineDNAwerespottedonamembrane(AmershamHybond-N+).Themembranewasincubatedwith2μg/mlofthemouse5-hydroxymethylcytosinemonoclonalantibody(dilution1:500).Themembraneswereexposedfor30seconds.Concentration:20µg/20µlFormulation:PBS(ph7.4)containing0.05%sodiumazideSpeciesReactivity:Human,mouse,broadrangeCrossReactivity:Specificfor5–hmc.Doesnotreactwith5–mcorunmodifiedcytosine.Purification:ProteinA/GpurifiedImmunogen:nucleotideFormat:AqueousbuffersolutionStorage/StABIlity:Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.Application(s):hMeDIP(2.5µg/IP)DB(2µg/ml)ELISA(1:500)IFNotes:Theoptimalantibodyconcentrationshouldbedeterminedbytheend–user.Warning(s):Avoidfreeze/thawcyclesScientificCategory:AntibodiesProductType:AntibodyDatashownislot-specific.Contactusforspecificinformationonotherlots.

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