Applications
ApplicationNotes
ValidatedApplications:
DB:0.2-1µg/ml
IHC:0.1-1µg/ml
ICC:0.5-2µg/ml
MeDIP:0.2-2µg/ml
Immunogen
BSA-conjugated5-hydroxymethylcytosine.
Buffer
PBSwith50%glycerol,1%BSA,and0.09%sodiumazide.
References
J.Reichmannetal."Dualspindleformationinzygoteskeepsparentalgenomesapartinearlymammalianembryos."bioRxiv(2017)10.1101/198275.
Siref,A.B.etal."Diagnosticutilityofdual5-hmC/Melan-Aimmunohistochemistryindifferentiatingnodalnevusfrommetastaticmelanoma:Aneffectivefirst-linetestforthework-upofsentinellymphnodespecimen."J.CutanPathol.(2019)10.1111/cup.13412.
Dotblotof5-Hydroxymethylcytosine(5-hmC)antibody(rAb).
Membranewaspre-spottedwith50,5,and0.5ng/dotofdoublestranded5-Hydroxymethylcytosine(5-hmC)DNA,5-Methylcytosine (5-mC)DNA,andunmethylatedDNA.Thepre-spottedmembranewasthenblottedwithantibodyat0.2µg/ml.
MeDIPof5-Hydroxymethylcytosine(5-hmC)antibody(rAb).
MeDIPwasperformedusing5-Hydroxymethylcytosineantibody(CloneRM236)ata10:1DNA:Abratio.1ngofunmethylated,5-Methylcytosine(5-mC)or5-Hydroxymethylcytosine(5-hmC)DNAstandard(897bp)wasspikedin1µgof genomicDNAisolatedfromHeLacellsasthecontrol.RealtimePCRwasthenperformedtodeterminethecaptureofDNAstandardasin%ofinput.
ImmunofluoresencestainingofHeLacellsusing5-Hydroxymethylcytosineantibody(CloneRM236)at0.5µg/mL(red).Actinfilamentsarelabeledwithfluoresceinphalloidin(green).HeLacellswerefixedwith4%paraformaldehydeandpermeABIlizedwithmethanol(−20°C)beforetreatmentwith2NHClfor30minat37°CtodenatureDNA.
Immunohistochemicalstainingofformalinfixed,paraffinembeddedhumanbraintissuesections,using5-Hydroxymethylcytosineantibody(CloneRM236)at1µg/ml.
Background
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(rAb).
Storage
Someproductsmaybeshippedatroomtemperature.Thiswillnotaffecttheirstabilityorperformance.Uponreceipt,unconjugatedantibodiesmaybestoredat-20°Cforupto2years.Fluorophore-&enzyme-conjugatedantibodiesshouldbestoredat4°C.Fluorophore-conjugatedantibodiesshouldbeprotectedfromlight.Keepreagentsonicewhennotinstorage;toavoidrepeatedfreeze/thawcycles,werecommendaliquotingitemsthatwillbestoredfrozenintosingle-usefractionspriortofreezing.
Guarantee
Thisproductisforresearchuseonlyandisnotforuseindiagnosticprocedures.Thisproductisguaranteedfor6monthsfromdateofarrival.
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
