AnhydroxymethylatedDNAIP(hMeDIP)wasperformedusingthemousemonoclonalantibodydirectedagainst5-hydroxymethylcytosine(Cat.#25202).
TheIgGisotypeantibodiesfrommousewereusedasnegativecontrol.TheDNAwaspreparedwiththeGenDNAmoduleofthehMeDIPkitandsonicatedtohaveDNAfragmentsof300-500bp.1μgofhumanHeLacellsDNAwerespikedwithnon-methylated,methylated,andhydroxymethylatedPCRfragments.TheimmunoprecipitatedmaterialwasanalyzedbyqPCRusingtheprimerpairspecificforthe3differentcontrolsequences.Theobtainedresultsshowthatthemousemonoclonalfor5-hmCishighlyspecificforthisbasemodification(noIPwithnon-methylatedormethylatedCbasescontainingfragments).
Determinationofthe5-hmCmousemonoclonalantibodytiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionofthemousemonoclonalantibodydirectedagainst5-hmC(Cat.#25202)inantigencoatedwells.TheantigenusedwasKHLcoupledto5-hmCbase.Byplottingtheabsorbanceagainsttheantibodydilution,thetiteroftheantibodywasestimatedtobe1:40,000.
Dotblotanalysisofthe5-hmCmousemonoclonalantibodywiththeC,mCandhmCPCRcontrols
200to2ng(equivalentof10to0.1pmolofC-bases)ofthehmC(1),mC(2)andC(3)PCRcontrolsfrom5-hmC,5-mC&cytosineDNAwerespottedonamembrane(AmershamHybond-N+).Themembranewasincubatedwith2μg/mlofthemouse5-hydroxymethylcytosinemonoclonalantibody(dilution1:500).Themembraneswereexposedfor30seconds.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
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