hMeDIPresultsobtainedwiththeantibodydirectedagainst5-hmC
hMeDIP(hydroxymethylatedDNAIP)wasperformedusingtheantibodyagainst5-hydroxymethylcytosine(Cat.No.25203).DNAfrommouseEScellswaspreparedwiththeGenDNAmoduleofthehMeDIPkitandsonicatedtoobtainDNAfragmentsof300-500bp.OneμgofshearedDNAwasspikedwithunmethylated(C)methylated(mC),andhydroxymethylated(hmC)controls.hMeDIPwasperformedwith3.5μgoftherabbit5-hmCantibodyandtheimmunoprecipitatedDNAwasanalysedbyqPCRusingprimersspecificforthe3differentcontrolsequences.Figure1showsthattherabbitpolyclonalantibodyagainst5-hmCishighlyspecificforthe5-hmCbasemodification(noIPwithnon-methylatedormethylatedCcontrolfragments).

Determinationoftheantibodytiter
Todeterminethetiter,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainst5-hmC(cat.No.25203),crudeserumandflowthrough,inantigencoatedwells.Theantigenusedwasthe5-hmCbasecoupledtoBSA.Byplottingtheabsorbanceagainsttheantibodydilution,thetiteroftheantibodywasestimatedtobe1:2,800.

Dotblotanalysisusingtheantibodydirectedagainst5-hmC
Todemonstratethespecificityoftheantibodyagainst5-hmC(cat.No.25203),aDotblotanalysiswasperformedusinghmC,mCandCcontrols.Onehundredto4ng(equivalentof5to0.2pmolofC-bases)ofthecontrolswerespottedonamembrane(AmershamHybond-N+).Theantibodywasusedatadilutionof1:1,000.Figure3showsahighspecificityoftheantibodyforthehydroxymethylatedcontrol.

Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.