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