首页 > Epigentek > Epigentek/MethylFlash Urine 5-Methylcytosine (5-mC) Quantification Kit (Colorimetric)/P-1039-96/96 assays

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Epigentek/MethylFlash Urine 5-Methylcytosine (5-mC) Quantification Kit (Colorimetric)/P-1039-96/96 assays

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P-1039-96

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InputType:UrineResearchArea:DNAMethylationTargetApplication:AmountQuantitationVesselFormat:96-WellPlate100%Guarantee:6monthsProductOverviewTheMethylFlash™Urine5-Methylcytosine(5-mC)QuantificationKit (Colorimetric) isacompletesetofoptimizedbuffersandreagentstocolorimetricallyquantify5-mCinurineusinganinhibitorycompetitiveimmunoassaymethod.It issuitablefordetectingtotalurinary5-methylcytosinelevels,resultingfromwholebodyturnoverordegradationofmethylatedDNA/RNA,inurinefromhumansandanimals.Theurinesamplescanbeinfreshorfrozenform. Thekithasthefollowingadvantagesandfeatures:Innovativecolorimetricassaywitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin4hours.96strip-wellmicroplateformatmakestheassayflexIBLe:manualorhighthroughputanalysis.Innovativekitcompositionenablesbackgroundsignalstobeextremelylow,whicheliminatestheneedforplateblockingandallowstheassaytobesimple,accurate,reliable,andconsistent.Thelevelof5-mCmeasuredinhumanurinesamplesusingthiskitiscomparabletothatdetectedbyHPLCmethod. Anovelassayprincipleallowshighsensitivitytobeachieved.Thedetectionlimitcanbeaslowas0.03ng/assaywellor1nMof5-mC.Lowinputrangeofurineforeachassaywithavolumeof0.5to5µlandanoptimalvolumeof1µl.Optimizedantibodyandenhancersolutionsallowforhighspecificityto5-mC,withoutcross-reactivitytounmethylatedcytosine. Negativecontrolandpositivestandardareincluded,whicharesuitableforquantificationof5-mCinfreeformand5-mCcontainedinmethylatedDNA/RNAfragmentsfromdifferenturinesamples.BackgroundInformationNucleobase5-methylcytosine(5-mC),amodifiedformofcytosineconvertedbycytosinemethyltransferases,iswidespreadbothinDNAanddifferentcellularRNAs.TheBIOLOGicalimportanceofDNA5-mCmethylationasamajorepigeneticmodificationinphenotypeandgeneexpressionhasbeenwidelyrecognized.RecentdatastronglysuggeststhatRNA5-mCmethylationisalsoinvolvedintheregulationofvariousbiologicalprocessesincludingtRNAstABIlity/recognitionandmRNAtranslation. Urinaryexcretionof5-mCincludingboth5-methyl-2-deoxycytidineand5-methylcytidineisanindicationofawholebodyturnoverordegradationofmethylatedDNAandRNA.Theurinary5-mClevelcanbealteredbyachangeofthebodies’turnoverofmethylatedDNA/RNAoralterationofcellularDNA/RNAmethylationstatus.Anumberofstudieshaveindicatedthat5-mCexcretedinurinehasthepotentialtoactasacancerbioMarker,withanincreasedlevelarisingfromdiseaseonsetandprogression.Forexample,anelevatedlevelofurinary5-mCwasobservedinlungcancer,breastcancer,andleukemiapatientswithactivediseasestates.Itwasalsoshownthaturinary5-mCexcretioniseffectedbyAlzheimer’sdiseaseorbyrADIationtreatment.Itissuggestedthaturinary5-mCmightbeapplicableasabiologicalmarkerfordetectingsometypesofcancerandmonitoringcancerprogressionaftertreatmentwithradiationordemethylationreagents. Chromatography-basedtechniquessuchasHPLCandTLCmassspectrometryarecommonlyusedfordetecting5-mCinurineandalthoughfairlyacurate,aretimeconsuming,lesssensitive,andhavelowthroughputwithhighcosts.Toaddressthisproblem,EpigentekofferstheMethylFlash™ Urine5-Methylcytosine(5-mC)QuantificationKit (Colorimetric) toquantify5-mCorbodyturnoverstatusofmethylatedDNA/RNAusingurinesamples.Principle&ProcedureInthisELISA-likeinhibitorycompetitiveimmunoassayassay,urinesamplesandthe5-mCstandardarefirstincubatedwitha5-mCantibodysolutionandthentransferredtostripwellscoatedwithmethylatedDNA(5-mC).Thewelliswashedtoremoveanyunboundreagentsafterincubationandthenadetectionantibodyisaddedtogenerateasignalthatcanbemeasuredcolorimetricallybyreadingtheabsorbanceinamicroplatespectrophotometer.Because5-mCintheurinesampleinhibitsthebindingof5-mCantibodyto5-mCcoatedonthewell,higherconcentrationsof5-mCintheurinesampleleadtoareducedbindingoftheantibodytothe5-mConthewell.ThereforethesignalorODintensitymeasuredfromthewellwillbeinverselyproportionaltotheamountof5-mCintheurinesampleandtheamountof5-mCintheurinesamplecanbequantifiedbyacomparisonwithapredetermined5-mCstandard.Fig.1.SchematicprocedurefortheMethylFlash™Urine5-MethylcytosineQuantificationKit(Colorimetric).Fig.2. 5-mCstandardwasaddedintotheassaywellsatdifferentconcentrationsandthenmeasuredwiththeMethylFlash™Urine5-MethylcytosineQuantificationKit (Colorimetric).Fig.3. 5-mClevelisquantifiedfromdifferentvolumesofhumanurineusingtheMethylFlash™Urine5-MethylcytosineQuantificationKit (Colorimetric). ProductComponentsWB(10XWashBuffer)BS(BindingSolution)MS(5-mCStandard,4µg/ml)*ME3(NegativeControl,50µg/ml)*ME4(5-mCDNAsolution,320X)*ME5(CaptureAntibody,1000X)*ME6(DetectionAntibody,2000X)*ES(EnhancerSolution)*DS(DeveloperSolution)SS(StopSolution)Plate1(AssayPlate)(WithFrame)Plate2(SamplePreparationPlate)UserGuide*Spinthesolutiondowntothebottompriortouse.

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