The Epigenase™m6ADemethylaseActivity/InhibitionAssayKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallymeasuretheactivity/inhibitionoftotalm6Ademethylasesusingnuclearextractsorpurifiedm6AdemethylaseslikeFTOandALKBH5fromabroadrangeofspeciessuchasmammalian,plant,fungal,andbacterial,inavarietyofformsincluding,butnotlimitedto,culturedcellsand,freshandfrozentissues.Thiskithasthefollowingadvantagesandfeatures:

• Colorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin5hours.
• Innovativekitcompositionenablesbackgroundsignalstobeextremelylowandallowstheassaytobesimple,accurate,reliable,andconsistent.
• Bothcell/tissuenuclearextractsandpurifiedproteinscanbeused,whichallowsdetectionofinhibitoryeffectsofm6Ademethylaseinhibitorinvivoandinvitro.
• Novelassayprincipleallowshighsensitivitytobeachieved.Theactivitycanbedetectedfromaslowas2µgofnuclearextractsor50ngofpurifiedenzymes.
• Theassaystandardisincluded,whichallowsthespecificactivityofm6Ademethylasetobequantified. 
• Strip-wellmicroplateformatmakestheassayflexIBLeformanualorhighthroughputanalysis.

BackgroundInformation 
N6-methyladenosine(m6A)isthemostcommonandabundantmodificationonRNAmoleculespresentineukaryotes.Recently,DNAm6AisalsoidentifiedinmulticellulareukaryotesincludingCaenorhaBDitiselegansandDrosophilamelanogaster,andfurThermoreidentifiedinhighereukaryotesincludingplants,mouseandhumancells.m6AplayscrucialrolesinregulatingDNAreplication,DNAdamage,RNAsplicing,transposition,transcription,andcellulardefense.Inhumancells,them6AmodificationisprobablycatalyzedbyamethyltransferasecomplexMETTL3/METTL14andremovedbytheα-ketoglutarate(α-KG)-andFe2+-dependentdioxygenasessuchasFTO,ALKBH5andTET-likeenzymes.ItwasshownthatMETTL3andα-KG/Fe2+-dependentdioxygenasesplayimportantrolesinmanyBIOLOGicalprocesses,rangingfromdevelopmentandmetabolismtofertility.Thedynamicandreversiblechemicalm6AmodificationonDNA/RNAmayalsoserveasanovelepigeneticMarkerofprofoundbiologicalsignificance.Down-regulationofm6Amodificationwasfirstcharacterizedinhumancancercellsandtissues,relativetotheirnormalcontrols.m6AisfoundtobethemostregulatedDNAmodificationincancers.Inadditiontotheregulationincancercells,relativetotheprimarycell/tissueswhichcontainquitelowDNAm6A(<0.001%),ahundreds-foldincreaseofm6Amodificationwasfoundforinvitroculturedhumancells(0.03%-0.22%). 

Principle&Procedure
Thiskitisdesignedformeasuringtotalm6Ademethylaseactivity/inhibition.Inanassaywiththiskit,theuniquem6Asubstrateisstablycoatedonthestripwells.Activem6Ademethylasesbindtoanddemethylatem6Acontainedinthesubstrate.Theun-demethylatedm6Ainthesubstratecanberecognizedwithahighaffinitym6Aantibodyandtheimmuno-signalisenhancedwithenhancersolution.Theratiooramountofun-demethylatedm6A,whichisinverselyproportionaltoenzymeactivity,canthenbecolorimetricallyquantifiedthroughanELISA-likereaction.

StartingMaterials
Inputmaterialscanbenuclearextractsorpurifiedenzymes.Theamountofnuclearextractsforeachassaycanbe2µgto20µgwithanoptimalrangeof5µgto10µg.Theamountofpurifiedm6ADNAdemethylasescanbe20ngto1µgwithanoptimalrangeof50ngto500ng,dependingonthepurityandcatalyticactivityoftheenzymes.

WB(10XWashBuffer)
DB(DemethylaseBuffer)
MS(10Xm6ASubstrate)*
BS(BindingSolution)
AS(AssayStandard,2µg/ml)*
CA(CaptureAntibody,1000µg/ml)*
DA(DetectionAntibody,400µg/ml)*
ES(EnhancerSolution)*
DS(DeveloperSolution)
SS(StopSolution)
Co-Factor1*
Co-Factor2*
Co-Factor3*
8-WellAssayStrips(WithFrame)

*Formaximumrecoveryoftheproducts,centrifugetheoriginalvialpriortoopeningthecap.