Applications

Methyl-DNA Immunoprecipitation ValidatedImmunofluorescence ValidatedDot Blot ValidatedImmunohistochemistry ValidatedImmunocytochemistry Validated

ApplicationNotes


ValidatedApplications:
MeDIP:1-2µgperIP
MeDIP-chip:10µgperIP
DB:0.2µg/mldilution

PublishedApplications:
MeDIP
ICC/IF
IHC(FFPE)

Seereferencesformoreinformation.Individualoptimizationmayberequired.

Immunogen

This5-Hydroxymethylcytosineantibodywasraisedagainst5-hydroxymethylcytidineconjugatedtoKLHandrecognizes5-hydroxymethylcytosine.

Buffer

PurifiedIgGin70mMTris(pH8),105mMNaCl,31mMglycine,0.07mMEDTA,30%glyceroland0.035%sodiumazide.Sodiumazideishighlytoxic.

References

5-Hydroxymethylcytosine (5-hmC) antibody (mAb) tested by hMeDIP-chip analysis.

hMeDIP-chipperformedonhumanbrainDNAusing5-Hydroxymethylcytosine(5-hmC)antibody.
HumanbrainDNA(2µg)wasimmunoprecipitatedwith10µgof5-Hydroxymethylcytosineantibody.FollowinghMeDIP,theDNAwasamplified,labeledandhybridizedtoanAffymetrixHumanTiling2.0RArray.Shownisaregionfromchromosome6qcontainingtheARID1B,ZDHHC14andSNX9genes.Theresultsshowthat5-hydroxymethylcytosineisenrichedprimarilyinthecodingregionsofgenes,ratherthaninthegenepromoterorregulatoryregions.

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

5-Hydroxymethylcytosine(5-hmC,5-hydroxymethylcytidine)antibodytestedbyMethylDNAimmunoprecipitation.
DNA(25pg)derivedfromthepromoteroftheAPCgenewasspikedinto500ngofhumangenomicDNAandsubjectedtotheMeDIPprocedureusing2μgof5-Hydroxymethylcytidineantibody(5hmC,maroonbars)or2μgofcontrolrabbitIgG(IgG,bluebars).RealtimequantitativePCRwasperformedontheimmunoprecipitatedDNAandresultsplottedas%ofinputDNA.ThespikedAPCDNAcontainedeithernomethylation(DNA),5-methylcytosinemethylation(5-mC)or5-hydroxymethylcytosinemethylation(5-hmC).

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

5-Hydroxymethylcytosine(5-hmC)antibody(mAb)testedbydotblotanalysis.
DNAsampleswerespotted(indicatedinngontheleft)ontoapositivelychargednylonmembraneandblottedwith5-Hydroxymethylcytidineantibodyata0.2µg/mldilution.

Lane1:double-strandedDNAcontaining5-hydroxymethylcytosine.
Lane2:double-strandedDNAcontaining5-methylcytosine.
Lane3:unmethylateddouble-strandedDNA.
Lane4:single-strandedDNAcontaining5-hydroxymethylcytosine.
Lane5:single-strandedDNAcontaining5-methylcytosine.
Lane6:unmethylatedsingle-strandedDNA.

Background

Inadditiontothismonoclonal,ActiveMotifofferstwopolyclonalantibodiesthatrecognize5-hydroxymethylcytosine,awholeserumversion(39769)andapurifiedIgGversion(39791).AllarevalidatedforuseinmethylDNAimmunoprecipitation(MeDIP).ForcustomersthatrequiretheABIlitytoquantitatetheamountofIgGintheMeDIPreaction,werecommendeitherthismonoclonalorthepurifiedIgGpolyclonal(39791).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.

BridgingAntibodytoImproveChIP,MeDIPandIP

SomeisotypesofmouseIgGantibodiesdonotbindwelltoproteinG-conjugatedagarose/magneticbeads,whicharecommonlyusedinChIP,MeDIPandIPexperiments.Captureefficiencycanbeimprovedbyincludingananti-mouseIgGbridgingantibodybecauseitbindswithastrongaffinitytoboththeproteinGbeadsandthemouseIgGantibody.ByincreasingtheaffinityofthemouseprimaryantibodyfortheproteinGbead,theBridgingAntibodyforMouseIgGcanhelpimprovetheresultsofallChIP,MeDIPandIPexperimentsthatuse5-Hydroxymethylcytosine(5-hmC)antibody(mAb).

CompatibleSecondaryAntibodies

ActiveMotifhasdevelopedavarietyofhigh-qualitysecondaryantibodiesformostapplications,withanti-rabbitandanti-mouseconjugatestoabroadlineofhigh-qualityfluorescentmolecules,aswellashorserADIshperoxidase(HRP).VisitourSecondaryAntibodyConjugatespagetofindtheperfectsecondariesforimmunofluorescenceexperimentswith5-Hydroxymethylcytosine(5-hmC)antibody(mAb).

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