首页 > Active Motif > Active Motif（艾跃）/5-Hydroxymethylcytosine (5-hmC) antibody (mAb)/40900/50µg

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Active Motif（艾跃）/5-Hydroxymethylcytosine (5-hmC) antibody (mAb)/40900/50µg

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40900

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ApplicationsApplicationNotesValidatedApplications:MeDIP:1-2µgperIPMeDIP-chip:10µgperIPDB:0.2µg/mldilutionPublishedApplications:MeDIPICC/IFIHC(FFPE)Seereferencesformoreinformation.Individualoptimizationmayberequired.ImmunogenThis5-Hydroxymethylcytosineantibodywasraisedagainst5-hydroxymethylcytidineconjugatedtoKLHandrecognizes5-hydroxymethylcytosine.BufferPurifiedIgGin70mMTris(pH8),105mMNaCl,31mMglycine,0.07mMEDTA,30%glyceroland0.035%sodiumazide.Sodiumazideishighlytoxic.ReferenceshMeDIP-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.BackgroundInadditiontothismonoclonal,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,MeDIPandIPSomeisotypesofmouseIgGantibodiesdonotbindwelltoproteinG-conjugatedagarose/magneticbeads,whicharecommonlyusedinChIP,MeDIPandIPexperiments.Captureefficiencycanbeimprovedbyincludingananti-mouseIgGbridgingantibodybecauseitbindswithastrongaffinitytoboththeproteinGbeadsandthemouseIgGantibody.ByincreasingtheaffinityofthemouseprimaryantibodyfortheproteinGbead,theBridgingAntibodyforMouseIgGcanhelpimprovetheresultsofallChIP,MeDIPandIPexperimentsthatuse5-Hydroxymethylcytosine(5-hmC)antibody(mAb).CompatibleSecondaryAntibodiesActiveMotifhasdevelopedavarietyofhigh-qualitysecondaryantibodiesformostapplications,withanti-rabbitandanti-mouseconjugatestoabroadlineofhigh-qualityfluorescentmolecules,aswellashorserADIshperoxidase(HRP).VisitourSecondaryAntibodyConjugatespagetofindtheperfectsecondariesforimmunofluorescenceexperimentswith5-Hydroxymethylcytosine(5-hmC)antibody(mAb).StorageSomeproductsmaybeshippedatroomtemperature.Thiswillnotaffecttheirstabilityorperformance.Uponreceipt,unconjugatedantibodiesmaybestoredat-20°Cforupto2years.Fluorophore-&enzyme-conjugatedantibodiesshouldbestoredat4°C.Fluorophore-conjugatedantibodiesshouldbeprotectedfromlight.Keepreagentsonicewhennotinstorage;toavoidrepeatedfreeze/thawcycles,werecommendaliquotingitemsthatwillbestoredfrozenintosingle-usefractionspriortofreezing.GuaranteeThisproductisguaranteedfor6monthsfromdateofreceipt.Thisproductisforresearchuseonlyandisnotforuseindiagnosticprocedures.ApplicationKeyChIP=ChromatinImmunoprecipitation;DB=DotBlot;ELISA=Enzyme-linkedImmunosorbentAssay;EMSA=ElectrophoreticMobilityShiftAssayFC=FlowCytometry;ICC=Immunocytochemistry;IF=Immunofluorescence;IHC=Immunohistochemistry;IP=Immunoprecipitation;MeDIP=Methyl-DNAImmunoprecipitation;TR-FRET=Time-ResolvedFluorescenceResonanceEnergyTransfer;WB=WesternBlotting

Active Motif 公司是一家致力于研发和生产核细胞分子生物学和表观遗传学研究用试剂的专业公司，也是世界上最大的转录因子试剂生产商和 ChIP-seq 外包服务供应商。Active Motif 的产品涉及：表观遗传学相关抗体、Chip 级别抗体、重组识别子和催化酶、预甲基化修饰的重组组蛋白、多聚核小体和生物素标记核小体；以及各类染色质分析、转录因子表达调控分析和 DNA 甲基化分析的试剂盒等。不仅如此，Active Motif 还为研究者提供专业的表观遗传学服务，特别是 Chip-seq 的外包服务。公司总部位于美国加州，目前在欧洲，日本，中国设有分公司。产品销售范围覆盖全球 30 多个国家和地区。