InputType: | DNA |
ResearchArea: | DNAMethylation |
TargetApplication: | AmountQuantitation |
VesselFormat: | 96-WellPlate |
100%Guarantee: | 6months |
The MethylFlash™5-Formylcytosine(5-fC)DNAQuantificationKit(Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantify5-formylcytosine(5-fC)inamicroplate-basedformat.ItissuitableforusewithDNAisolatedfromanyspecies(mammals,plants,fungi,bacteria,viruses,etc.)inavarietyofformsincluding,butnotlimitedto,culturedcells,freshandfrozentissues,paraffin-embeddedtissues,andbodyfluidsamples.Thekithasthefollowingadvantagesandfeatures:
BackgroundInformation
5-formylcytosine(5-fC)hasrecentlybeenfoundinmammaliantissueandcellsandhasbeenclassifiedasaseventhDNAbase.Itisformedbytheoxidationof5-hydroxymethylcytosinethroughTEThydroxylasesandhasbeendemonstratedtobeanintermediateoftheDNAdemethylationprocess.Thefunctionof5-fCingeneregulationisnotyetentirelyclear,butithasbeenshownthat5-fCexhibitsreplication-dependentdilutionduringmousepre-implantationdevelopmentandcouldbefunctionalinregulatingpre-implantationdevelopmentasawhole.Thedetectionof5-fCinvarioustissuesandcellsisimportantbecause5-fCcouldbeaMarkertoindicateactiveDNAdemethylation.5-fCcanalsobedirectlyexcisedbyThymineDNAglycosylase(TDG)toallowsubsequentbaseexcisionrepair(BER)processingwhichconvertsmodifiedcytosinebacktoitsunmodifiedstate.
Severalchromatography-basedtechniquessuchasHPLC-MSareusedforthedetectionof5-fCintissueandcells.However,thesemethodsaretimeconsuming,requirelargeamountsofDNAandhavelowthroughputwithhighcosts.Toaddresstheseproblems,EpigentekofferstheMethylFlash™5-Formylcytosine(5-fC)DNAQuantificationKit(Colorimetric),whichusesauniquemicroplate-basedproceduretodirectlyquantify5-fC.
Principle&Procedure
TheMethylFlash™5-Formylcytosine(5-fC)DNAQuantificationKit(Colorimetric)containsallthereagentsnecessaryforthequantificationof5-fC.Inthisassay,DNAisboundtostrip-wellsthatarespecificallytreatedtohaveahighDNAaffinity.5-fCisdetectedusingcaptureanddetectionantibodies.ThedetectedsignalisenhancedandthenquantifiedcolorimetricallybyreADIngtheabsorbanceinamicroplatespectrophotometeratawavelengthof450nm.Theamountof5-fCisproportionaltotheODintensitymeasured.
StartingMaterials&InputAmount
ThedetectionlimitoftheinputDNAcanbeaslowas1pgof5-fCwithadynamicrangefrom10pgto400pg(seeFig.2).Thereisnocross-reactivitytocytosine,5-mC,or5-hmCwithintheindicatedconcentrationrangeofthesampleDNA(seeFig.3).
SafeandConvenient
Allthenecessaryreagents,includingnegativeandpositivecontrols,forthequantificationof5-fCareconvenientlypackagedinthekit.ThedirectcolorimetricquantificationofDNAsamplesina96-wellmicroplateformateliminatestheneedforDNAdigestion/denaturation,radioactivity,extraction,orchromatography.
Easy,Fast,andFlexible
Theentirecolorimetricassayhaseasy-to-followstepsforconvenienceandspeed,allowingittobecompletedinjust3hoursand45minutes.Thestrip-wellmicroplateformatallowsforaflexibleassayformanualorhighthroughputanalysis.Universalpositiveandnegativecontrolsareincludedwiththekitforthequantificationof5-fCfromanyspecies,suchasmammals,plants,fungi,bacteria,andviruses.
Responsive,Reliable,andPractical
Basedonitsworkingprincipleandthemicroplateformat,thekitcanbepracticallyandroutinelyusedforanyspeciesandforavarietyofformsincludingculturedcells,freshandfrozentissues,andparaffin-embeddedtissues.TodemonstratethecapABIlitiesofthekit,ithasbeensuccessfullyusedforquantifyingthecontentof5-fCinDNAfromhumanbraintissue, mousebraintissue, rectaltissue,andrectalcancer.Thepercentageof5-fCmeasuredbythekitisparallelandcomparabletothatdetectedbyLC-MS/MSmethods(seeFig.4).
WB(10XWashBuffer)
BS(BindingSolution)
NC(NegativeControl,10µg/ml)*
PC(PositiveControl,5-fC1µg/ml)*
CA(CaptureAntibody,1000X)*
DA(DetectionAntibody,2000X)*
ES(EnhancerSolution)*
DS(DeveloperSolution)
SS(StopSolution)
8-WellAssayStrips(WithFrame)
UserGuide
*Spinthesolutiondowntothebottompriortouse.
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