TheEpiQuik™DNADemethylaseActivity/InhibitionAssayUltraKitisacompletesetofessentialcomponentswhichenablesanexperimentertomeasuretotalDNAdemethylaseactivity/inhibition.Thekitcanbeusedwithnuclearextractsfromabroadrangeofspeciessuchasmammalsandplants,inavarietyofformsincluding,butnotlimitedto,culturedcellsandfreshtissues. TheEpiQuik™DNADemethylaseActivity/InhibitionAssayUltraKit hasthefollowingfeatures: - Colorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbecompletedwithin4hours.
- SafeandinnovativecolorimetricassaywithouttheneedforrADIoactivity,extraction,orchromatography.
- Anultra-sensitivedetectionlimit,aslowas1µgofnuclearextract,whichisfivetimesmoresensitivethanthepredecessorkit.
- 96strip-wellmicroplateformatallowsforeitherloworhighthroughputanalysis.
BackgroundInformation
DNAdemethylationisnecessaryfortheepigenenticreprogrammingofgenesandisalsodirectlyinvolvedinmanyimportantdiseasemechanismssuchastumorprogression.DemethylationofDNAcaneitherbepassiveoractive,oracombinationofboth.PassiveDNAdemethylationusuallytakesplaceonnewlysynthesizedDNAstrandsviaDNMT1duringreplicationrounds.ActiveDNAdemethylationmainlyoccursbytheremovalof5-methylcytosinethroughfurthermodifiedcytosinebaseswhichhavebeenconvertedbyTETenzyme-mediatedoxidation.TheseoxidationproductshavebeenshowntoberepairedbyTDG,aglycosylasewhichisinvolvedinbaseexcisionrepair,ordirectlyconvertedtocytosinebyDNMT3A/DNMT3Binoxidativestates.ItisproposedthatDNAdemethylationcouldalsobeinitiatedbydeaminationof5-mCthroughcandidatedeaminasesincludingAIDandAPOBEC1,whichconvert5-mCtothymine.TheresultingthyminecouldberepairedbyBERinitiatedbyaT-GmismatchglycosylasesuchasMBD4orTDG.Inaddition,the5-mCbasecanbedirectlyremovedinplantsbytheDME/ROS1familyof5-mCDNAglycosylases,resultinginanabasicsitethatisrepairedbytheBERprocess. Principle&Procedure
Inanassaywiththiskit,theuniquemethylatedDNAsubstrateisstablycapturedonthestripwells.ActiveDNAdemethylasesbindtoanddemethylatetheDNAsubstrate.ThemethylatedDNAcanberecognizedwithahighaffinity5-methylcytosineantibodyandtheimmuno-signalisenhancedwithenhancersolution.TheratiooramountofmethylatedDNA,whichisinverselyproportionaltoenzymeactivity,canthenbecolorimetricallyquantifiedthroughanELISA-likereaction. StartingMaterials&InputAmount
Theamountofnuclearextractsforeachassaycanbefrom2µgto20µgwithanoptimalrangeof5to10µg. WerecommendusingEpigentek"snuclearextractionkitforoptimalresults. | 
 Fig.1.SchematicprocedureoftheEpiQuik™DNADemethylaseActivity/InhibitionAssayUltraKit.
Fig.2. AssaycontrolstandardwasaddedintotheassaywellsatdifferentconcentrationsandthenmeasuredwiththeDNADemethylaseActivity/InhibitionAssayUltraKit.
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