Determinationofthe5-hmCrabbitpolyclonalantibodytiter
Todeterminethetiter,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,mCandhmCPCRcontrols
100to4ng(equivalentof5to0.2pmolofC-bases)ofthehmC,mCandCPCRcontrolswerespottedonamembrane(AmershamHybond-N+).Themembranewasincubatedwiththerabbit5-hydroxymethylcytosinepolyclonalantibody(dilution1:200).Themembraneswereexposedfor30seconds.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
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