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Dotblotanalysisusingtheantibodydirectedagainst5-caC
Todemonstratethespecificityoftheantibodyagainst5-caC(Cat.#25201),aDotBlotanalysiswasperformedusingsyntheticoligonucleotidescontainingdifferentmodifiedC-bases(indicatedinred).125and25ngoftherespectiveoligoswereboundtoaStreptavindin-coatedmulti-wellplate.Theantibodywasusedatadilutionof1:1,000.ThebindingofantibodytotheDNAwasmeasuredbyECLchemiluminescence.Figure1showsahighspecificityoftheantibodyforthecarboxylatedcytosine.
Immunofluorescenceassayusingtheantibodydirectedagainst5-caC
293TcellsweretransfectedwitheitherthemouseFLAG-taggedwild-typeTet1(Tet1CD)orthecatalyticallyinactiveFLAG-taggedC-terminaldomainofTet1(Tet1mCD)andstainedwiththeantibodyagainst5-caC(Cat.#25201),diluted1:500,andwithananti-FLAGantibody,followedbyDAPIcounterstaining.
Immunoprecipitationusingtheantibodydirectedagainst5-caC
Immunoprecipitationwasperformedwiththeantibodyagainst5-caC(Cat.#25201)on2µgofJ1ESgenomicDNA,spikedwith1pgofacontrolDNAfragment(approximately700bpfromtheRFP(Ringfingerprotein)gene)containingdifferentcytosinemodifications.ThemCandhmCcontrolDNAwasgeneratedbyPCRwiththecorrespondingnucleotide.ThecaCcontrolfragmentwasobtainedbyinvitromethylationusingM.SssImethyltransferasefollowedbyoxidationwithpurifiedTet2.TheimmunoprecipitatedDNAwassubsequentlyanaysedbyqPCRusingprimersspecificforthecontrolDNAfragmentsandforGAPDH,usedasanegativecontrol.Figure3showstheenrichmentcalculatedastheratiooftherecoveryofthecontrolDNAversustherecoveryoftheGAPDHnegativecontrol.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
IF(1:500)
DNAIP(4µggenomicDNA/IP)