TheEpigenase™ 5mC-HydroxylaseTETActivity/InhibitionAssayKit(Colorimetric)isacompletesetofoptimizedbuffersandreagentsformeasuringactivity/inhibitionoftotalcytosineoxygenaseTETenzymesinnuclearextractsorpurifiedTETisoforms(TETs1-3)fromabroadrangeofspeciessuchasmammals,plants,fungi,andbacteria,andinavarietyofformsincluding,butnotlimitedtoculturedcellsandfreshandfrozentissues.  Thekithasthefollowingadvantagesandfeatures:

• 5hourcolorimetricassayprocedurewitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin5hours.
• DirectlymeasuresTEThydroxylaseactivityviaastraightforwarddetectionofTET-convertedhydroxymethylatedproducts.
• Innovativekitcompositionenablesbackgroundsignalstobeextremelylowandallowstheassaytobesimple,accurate,reliable,andconsistent.
• Eithercell/tissueextractsorpurifiedTETproteinscanbeused,whichallowsfordetectionofinhibitoryeffectsofTEThydroxylaseinhibitors invivo and invitro.
• Novelassayprincipleallowshighsensitivitytobeachieved.Theactivitycanbedetectedfromaslowas20ngofpurifiedTET1hydroxylase.
• Ahydroxymethylatedstandardisincluded,whichallowsthespecificactivityofTEThydroxylasestobequantified. 
• StripmicroplateformatmakestheassayflexIBLeformanualorhighthroughputanalysis(96assays).

BackgroundInformation
5-hydroxymethylcytosine,asasixthDNAbasewithfunctionsintranscriptionregulation,hasbeendetectedtobeabundantinthehumanandmousebrainandembryonicstem(ES)cells.Inmammals,itcanbegeneratedbyoxidationof5-methylcytosine,areactionmediatedbytheten-eleventranslocation(TET)familyofcytosineoxygenases. 

TheTETfamilyofcytosineoxygenasesincludesTET1,TET2,andTET3.TheseTETproteinsmaypromoteDNAdemethylationbybindingtoCpG-richregionstopreventunwanted DNAmethyltransferase activity,andbyconverting5mCto5hmCandfurtherto5caC(5-carboxylcytosine)throughhydroxylaseactivity.Itwasshownthatgenomic5hmClevelscorrelatewithTEThydroxylaseactivity.Inaddition, TET1wasshowntohavedualfunctionsintranscriptionactivationandrepressionbybindingdifferenttargetgenesinEScells.TET1isalsoafusionpartneroftheMLLgeneinacutemyeloidleukemiaandisconsideredanoncoprotein.TET2isfoundtobefrequentlymutatedinleukemiaandconsideredtoactastumorsuppressor.TET3wasdemonstratedtoplayauniqueroleinDNAmethylationreprogrammingprocessesinthemammalianzygote.Thus,activatingtumorsuppressorTETenzymessuchasTET2orinhibitingoncoproteinTETenzymessuchasTET1wouldbeimportantinbenefitingcancerdiagnositcsanddevelopingnewtarget-basedcancertherapeutics.

Principle&Procedure
Inthisassay,amethylatedsubstrateisstablycoatedontothemicroplatewells.ActiveTETs(notincluded)bindtothesubstrateandconvertthemethylatedsubstratetohydroxymethylatedproducts.TheTET-convertedhydroxymethylatedproductscanberecognizedwithaspecificantibody.Theratiooramountofhydroxymethylatedproducts,whichisproportionaltoenzymeactivity,canthenbecolorimetricallymeasuredbyreADIngtheabsorbanceinamicroplatespectrophotometeratawavelengthof450nm.TheactivityoftheTETenzymeisinturnproportionaltotheopticaldensityintensitymeasured.

WB(10XWashBuffer)
TAB(TETAssayBuffer)
TS(10XTETSubstrate)*
BS(BindingSolution)
HC5(TETAssayStandard,20µg/ml)*
HC6(CaptureAntibody,1000µg/ml*
HC7(DetectionAntibody,400µg/ml)*
HC8(EnhancerSolution)*
DS(DeveloperSolution)
SS(StopSolution)
Co-factor1*
Co-factor2*
Co-factor3*
8-WellAssayStrips(WithFrame)

*Formaximumrecoveryoftheproducts,centrifugetheoriginalvialpriortoopeningthecap.