The MethylFlash™m6ADNAMethylationELISAKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentsto colorimetricallyquantifyN6-methyladenosine(m6Aor6mA)inDNA.Thekitissuitablefordirectlydetecting m6ADNAmethylationstatususingDNAisolatedfromanyspeciessuchasmammals,plants,fungi,bacteria,andviruses. Thekithasthefollowingadvantagesandfeatures:

• Colorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbecompletedwithin3hoursand45minutes.
• Highsensitivity,ofwhichthedetectionlimitcanbeaslowas5pgofm6A.
• UniquebindingsolutionallowsDNA>100ntstobetightlyboundtothewells,whichenablesquantificationofm6AfrombothintactDNAorfragmentedDNAsuchasChIPedDNA.
• Optimizedantibodyandenhancersolutionsallowhighspecificitytom6A,withnocross-reactivitytounmethylatedadenosinewithintheindicatedconcentrationrangeofthesampleDNA.
• Universalpositiveandnegativecontrolsareincluded,whicharesuitableforquantifyingm6Afromanyspecies.
• Strip-wellmicroplateformatmakestheassayflexIBLeformanualorhighthroughputanalysis.
• Simple,reliable,andconsistentassayconditions.

BackgroundInformation 
N6-methyladenosine(m6A)isthemostcommonandabundantmodificationonRNAmoleculespresentineukaryotes.DNAm6AisalsoidentifiedinmulticellulareukaryotesincludingCaenorhaBDitiselegansandDrosophilamelanogaster,andfurThermoreidentifiedinhighereukaryotesincludingplants,mouseandhumancells.m6AplayscrucialrolesinregulatingDNAreplication,transposition,transcription,andcellulardefense.Inhumans,theDNAm6AmodificationismostlikelycatalyzedbyamethyltransferasecomplexMETTL3andremovedbytheα-ketoglutarate(α-KG)-andFe2+-dependentdioxygenasessuchasALKBH5andTET-likeenzymes.ItwasshownthatMETTL3andα-KG/Fe2+-dependentdioxygenasesplayimportantrolesinmanyBIOLOGicalprocesses,rangingfromdevelopmentandmetabolismtofertility.Thedynamicandreversiblechemicalm6AmodificationonDNAmayalsoserveasanovelepigeneticMarkerofprofoundbiologicalsignificance.Down-regulationofm6Amodificationwasfirstcharacterizedinhumancancercellsandtissues,relativetotheirnormalcontrols. m6AisfoundtobethemostregulatedDNAmodificationincancers.Inadditiontotheregulationincancercells,relativetotheprimarycell/tissueswhichcontainquitelowamountsofDNAm6A(<0.001%),ahundreds-foldincreaseofm6Amodificationwasfoundforinvitroculturedhumancells(0.03%-0.22%).Therefore,identifyingm6ADNAmethylationlevelsanddistributiononDNAcouldadvanceunderstandingofepigeneticregulationofbiologicalprocessatthegenomiclevel,andfurtherprovideusefulinformationforimprovingdiagnosticsandtherapeuticsofdisease.  

Principle&Procedure
Thiskitcontainsallreagentsnecessaryforthequantificationofm6AinDNA.Inthisassay,DNAisboundtostripwellsusingDNAhighbindingsolution.m6Aisdetectedusingcaptureanddetectionantibodies.ThedetectedsignalisenhancedandthenquantifiedcolorimetricallybyreADIngtheabsorbanceinamicroplatespectrophotometer.Theamountofm6AisproportionaltotheODintensitymeasured. 

StartingMaterials
Startingmaterialscanincludevarioustissueorcellsamplessuchascellsfromflaskormicroplateculturedcells,freshandfrozentissues,paraffin-embeddedtissues,blood,bodyfluidsamples,etc.

WB(10XWashBuffer)
BS(BindingSolution)
NDC(NegativeDNAControl,100µg/ml)*
PC(PositiveControl, 200µg/mlcontainingm6A1µg/ml)*
CA(CaptureAntibody,1000X)*
DA(DetectionAntibody,1000X)*
ES(EnhancerSolution)*
DS(DeveloperSolution)
SS(StopSolution)
8-WellAssayStrips(WithFrame)

*Spinthesolutiondowntothebottompriortouse.