Applications

Methyl-DNA Immunoprecipitation ValidatedImmunofluorescence ValidatedDot Blot ValidatedImmunohistochemistry Validated

ApplicationNotes


ValidatedApplications:
MeDIP:1-2µgperIP
IHC(FFPE):1µg/mldilution
DB:0.2µg/mldilution

PublishedApplications:
MeDIP-Seq
MeDIP
IHC
IF

Seereferencesformoreinformation.Individualoptimizationmayberequired.

Immunogen

This5-Hydroxymethylcytidineantibodywasraisedagainst5-hydroxymethylcytidineconjugatedtoKLHandrecognizes5-hydroxymethylcytosine.

Buffer

PBSpH7.5containing30%glyceroland0.035%sodiumazide.Sodiumazideishighlytoxic.

References

5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by MeDIP analysis.

5-HydroxymethylcytidineantibodytestedbyMethylDNAimmunoprecipitation.
DNA(25pg)derivedfromthepromoteroftheAPCgenewasspikedinto500ngofhumangenomicDNAandsubjectedtotheMeDIPprocedureusing2μgof5-Hydroxymethylcytidineantibody(5hmC,maroonbars)or2μgofcontrolrabbitIgG(IgG,bluebars).RealtimequantitativePCRwasperformedontheimmunoprecipitatedDNAandresultsplottedas%ofinputDNA.ThespikedAPCDNAcontainedeithernomethylation(DNA),5-methylcytosinemethylation(5-mC)or5-hydroxymethylcytosinemethylation(5-hmC).The5-hmCantibodyshowsa650-foldenrichmentfor5-hmCDNAcomparedto5-mCDNAorunmethylatedDNA.

5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by dot blot analysis.

5-Hydroxymethylcytidineantibodytestedbydotblotanalysis.
DNAfromtheMethylatedDNAStandardKit(CatalogNo.55008)werespotted(indicatedinngontheleft)ontoapositivelychargednylonmembraneandblottedwith5-Hydroxymethylcytidineantibodyrecognizing5-hydroxymethylcytosine(1µg/mldilution).

Lane1:double-strandedDNA5-hydroxymethylcytosine.
Lane2:double-strandedDNAcontaining5-methylcytosine.
Lane3:doublestrandedunmethylatedDNA.
Lane4:single-strandedDNAcontaining5-hydroxymethylcytosine.
Lane5:single-strandedDNAcontaining5-methylcytosine.
Lane6:singlestrandedunmethylatedDNA.

5-Hydroxymethylcytosine (5-hmC) antibody (pAb) tested by immunohistochemistry.

5-Hydroxymethylcytosine(5-hmc)antibody(pAb)testedbyImmunohistochemistry
PunctatenuclearstainingpatternisdetectedinFormalin-fixed,paraffin-embeddedtissuesectionsfromhumansubstantianigra(midbrain).TopPanel:5-mCantibodyat1:1000dilution.BottomPanel:Noprimaryantibody(2ndstepantibodyalone)

Background

ActiveMotifofferstwopolyclonalantibodiesthatrecognize5-hydroxymethylcytosine,awholeserumversion(39769)andapurifiedIgGversion(39791).BotharevalidatedforuseinmethylDNAimmunoprecipitation(MeDIP).ForcustomersthatrequiretheABIlitytoquantitatetheamountofIgGintheMeDIPreaction,thepurifiedIgGversion(39791)isrecommended.Thewholeserumversion(39769)isveryhightitreandshouldbeusedcarefully(0.1-0.5µlperIP)asnottogeneratehighnon-specificbackground.Thewholeserumversion(39769)hasbeenusedsuccessfullyinimmunofluorescence(IF,Itoetal,2010),andthepurifiedIgGversion(39791)islikelytoworkinthisapplicationaswell.

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

Someproductsmaybeshippedatroomtemperature.Thiswillnotaffecttheirstabilityorperformance.Uponreceipt,unconjugatedantibodiesmaybestoredat-20°Cforupto2years.Fluorophore-&enzyme-conjugatedantibodiesshouldbestoredat4°C.Fluorophore-conjugatedantibodiesshouldbeprotectedfromlight.Keepreagentsonicewhennotinstorage;toavoidrepeatedfreeze/thawcycles,werecommendaliquotingitemsthatwillbestoredfrozenintosingle-usefractionspriortofreezing.

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