Applications

Immunofluorescence ValidatedDot Blot ValidatedImmunohistochemistry Validated

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.

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

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.

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

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. 

5-Hydroxymethylcytosine (5-hmC) antibody (rAb) tested by immunofluorescence.

ImmunofluoresencestainingofHeLacellsusing5-Hydroxymethylcytosineantibody(CloneRM236)at0.5µg/mL(red).Actinfilamentsarelabeledwithfluoresceinphalloidin(green).HeLacellswerefixedwith4%paraformaldehydeandpermeABIlizedwithmethanol(−20°C)beforetreatmentwith2NHClfor30minat37°CtodenatureDNA.

5-Hydroxymethylcytosine (5-hmC) antibody (rAb) tested by immunohistochemistry.

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