Applications
ApplicationNotes
ValidatedApplications:
MeDIP:0.1-0.5µlperIP
DB:1:10,000dilution
PublishedApplications:
MeDIP
MeDIP-Seq
ICC/IF
IHC(FFPE)
FC
DB
Seereferencesformoreinformation.Individualoptimizationmayberequired.
Immunogen
This5-Hydroxymethylcytosineantibodywasraisedagainst5-hydroxymethylcytidineconjugatedtoKLHandrecognizes5-hydroxymethylcytosine.
Buffer
Rabbitserumcontaining30%glyceroland0.035%sodiumazide.Sodiumazideishighlytoxic.
References
5-Hydroxymethylcytosine(5-hmC,5-hydroxymethylcytidine)antibodytestedbyMethylDNAimmunoprecipitation.
DNA(25pg)derivedfromthepromoteroftheAPCgenewasspikedinto500ngofhumangenomicDNAandsubjectedtotheMeDIPprocedureusing1μlof5-Hydroxymethylcytidineantibody(5hmC,maroonbars)or1μlofcontrolrabbitIgG(IgG,bluebars).RealtimequantitativePCRwasperformedontheimmunoprecipitatedDNAandresultsplottedas%ofinputDNA.ThespikedAPCDNAcontainedeithernomethylation(DNA),5-methylcytosinemethylation(5-mC)or5-hydroxymethylcytosinemethylation(5-hmC).
5-Hydroxymethylcytosine(5-hmC,5-hydroxymethylcytidine)antibodytestedbydotblotanalysis.
DNAsamples(10ngor50ngasindicated)werespottedontopositivelychargednylonmembraneandblottedwith5-Hydroxymethylcytidineantibodyatadilutionof1:10,000.
TopPanel:DoublestrandedDNA.
BottomPanel:SinglestrandedDNA.
Lane1:UnmethylatedDNA.
Lane2:DNAcontaining5-methylcytosine.
Lane3:DNAcontaining5-hydroxymethylcytosine.
5-Hydroxymethylcytosine(5-hmC,5-hydroxymethylcytidine)antibodytestedbydotblotanalysis.
DNAsampleswerespottedontopositivelychargednylonmembraneandblottedwithantibodiesasindicated.
PanelA:5-Hydroxymethylcytidineantibodyrecognizing5-hydroxymethylcytosine(1:10,000dilution).
PanelB:5-Methylcytidineantibody(1:1,000dilution).
Lane1:DNAderivedfrommouseembryonicstemcells(150ng).
Lane2:DNAderivedfrommousespleen(600ng).
Lane3:27baseoligonucleotidecontaining5-hydroxymethylcytosine(1.2ng).
Lane4:33baseoligonucleotidecontaining5-methylcytosine(2000ng).
Background
ActiveMotifofferstwopolyclonalantibodiesthatrecognize5-hydroxymethylcytosine,awholeserumversion(39769)andapurifiedIgGversion(39791).BotharevalidatedforuseinmethylDNAimmunoprecipitation(MeDIP).ForcustomerswhomustquantitatetheamountofIgGintheMeDIPreaction,thepurifiedIgGversion(39791)isrecommended.Thewholeserumversion(39769)isveryhightitre,soshouldbeusedcarefully(0.1-0.5µlperIP)topreventnon-specificbackground.Thewholeserumversion(39769)hasbeenusedsuccessfullyinimmunofluorescence(IF,Itoetal,2010);thepurifiedIgGversion(39791)islikelytoworkinIFaswell.
DNAmethylationisanepigeneticeventinwhichDNAmethyltransferases(DNMTs)catalyzethereactionofamethylgrouptothefifthcarbonofcytosineinaCpGdinucleotide.Thismodificationhelpstocontrolgeneexpressionandisalsoinvolvedingenomicimprinting,whileaberrantDNAmethylationisoftenassociatedwithdisease.5-methylcytosineisamodifiedbasethatisfoundintheDNAofplantsandvertebrates.
AsecondtypeofDNAmethylationexists,5-hydroxymethylcytosine(5-hydroxymethylcytosine,5-hmC).Thisresultsfromtheenzymaticconversionof5-methylcytosineinto5-hydroxymethylcytosinebytheTETfamilyofcytosineoxygenases.Thisantibodywasdevelopedspecificallytodistinguish5-hydroxymethylcytosinefrom5-methylcytosineasconventionalmethods(enrichmentbyantibodyormethylDNAbindingprotein,enzymaticdigestionandbisulfitesequencing)cannotdoso.ItispossIBLethat5-hydroxymethylcytosine(5-hmC)representsapathwaytodemethylateDNA,as5-hydroxymethylcytosineisrepairedasmismatchedDNAandreplacedwithunmethylatedcytosine.
CompatibleSecondaryAntibodies
ActiveMotifhasdevelopedavarietyofhigh-qualitysecondaryantibodiesformostapplications,withanti-rabbitandanti-mouseconjugatestoabroadlineofhigh-qualityfluorescentmolecules,aswellashorserADIshperoxidase(HRP).VisitourSecondaryAntibodyConjugatespagetofindtheperfectsecondariesforimmunofluorescenceexperimentswith5-Hydroxymethylcytosine(5-hmC)antibody(pAb).
Storage
Antibodiesinsolutioncanbestoredat-20°Cfor2years.Avoidrepeatedfreeze/thawcyclesandkeeponicewhennotinstorage.
Guarantee
Thisproductisguaranteedfor6monthsfromdateofreceipt.
Thisproductisforresearchuseonlyandisnotforuseindiagnosticprocedures.
ApplicationKey
- ChIP=ChromatinImmunoprecipitation;
- DB=DotBlot;
- ELISA=Enzyme-linkedImmunosorbentAssay;
- EMSA=ElectrophoreticMobilityShiftAssay
- FC=FlowCytometry;
- ICC=Immunocytochemistry;
- IF=Immunofluorescence;
- IHC=Immunohistochemistry;
- IP=Immunoprecipitation;
- MeDIP=Methyl-DNAImmunoprecipitation;
- TR-FRET=Time-ResolvedFluorescenceResonanceEnergyTransfer;
- WB=WesternBlotting
