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- 细胞P300/CBP组蛋白乙酰转移酶(P300/CBPHAT)活性检测试剂盒(比色)
Applications




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-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-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)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


