Applications

Methyl-DNA Immunoprecipitation ValidatedImmunofluorescence ValidatedDot Blot ValidatedImmunohistochemistry ValidatedFlow Cytometry ValidatedImmunocytochemistry Validated

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) antibody (pAb) tested by MeDIP analysis.

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) antibody (pAb) tested by dot blot analysis.

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) antibody (pAb) tested by dot blot analysis.

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