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

• 5hourfluorometricassayprocedurewitheasy-to-followstepsforconvenienceandspeed.Theentireprocedurecanbefinishedwithin5hours.
• DirectlymeasuresTEThydroxylaseactivityviaastraightforwarddetectionofTET-convertedhydroxymethylatedproducts.
• Innovativekitcompositionenablesbackgroundsignalstobeextremelylowandallowstheassaytobesimple,accurate,reliable,andconsistent.
• Eithercell/tissueextractsorpurifiedTETproteinscanbeused,whichallowsfordetectionofinhibitoryeffectsofTEThydroxylaseinhibitorsinvivo and invitro.
• Novelassayprincipleallowshighsensitivitytobeachieved.Theactivitycanbedetectedfromaslowas10ngofpurifiedTET1hydroxylase.
• Ahydroxymethylatedstandardisincluded,whichallowsthespecificactivityofTEThydroxylasestobequantified. 
• StripmicroplateformatmakestheassayflexIBLeformanualorhighthroughputanalysis(96assays).

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

TheTETfamilyofcytosineoxygenasesincludesTET1,TET2,andTET3.TheseTETproteinsmaypromoteDNAdemethylationbybindingtoCpG-richregionstopreventunwantedDNAmethyltransferaseactivity,andbyconverting5mCto5hmC andfurtherto5caC(5-carboxylcytosine) throughhydroxylaseactivity.Itwasshownthatgenomic5hmClevelscorrelatewithTEThydroxylaseactivity.Inaddition, TET1wasshowntohavedualfunctionsintranscriptionactivationandrepressionbybindingdifferenttargetgenesinEScells.TET1isalsoafusionpartneroftheMLLgeneinacutemyeloidleukemiaandisconsideredanoncoprotein.TET2isfoundtobefrequentlymutatedinleukemiaandconsideredtoactastumorsuppressor.TET3wasdemonstratedtoplayauniqueroleinDNAmethylationreprogrammingprocessesinthemammalianzygote.Thus,activatingtumorsuppressorTETenzymessuchasTET2orinhibitingoncoproteinTETenzymessuchasTET1wouldbeimportantinbenefitingcancerdiagnositcsanddevelopingnewtarget-basedcancertherapeutics.

Principle&Procedure
Inthisassay,amethylatedsubstrateisstablycoatedontothemicroplatewells.ActiveTETs(notincluded)bindtothesubstrateandconvertmethylatedsubstratehydroxymethylatedproductsfromthesubstrate.TheTET-convertedhydroxymethylatedproductscanberecognizedwithaspecificantibody.Theratiooramountofhydroxymethylatedproducts,whichisproportionaltoenzymeactivity,canthenbefluorometricallymeasuredbyreADIngthefluorescenceinafluorescentmicroplatereaderat530excitationand590emission.TheactivityoftheTETenzymeisinturnproportionaltotherelativefluorescentunitsmeasured.

Fig.2. DemonstrationofhighsensitivityandspecificityoftheTET1activity/inhibitionassayachievedbyusingrecombinantTET1withtheEpigenase5-mCHydroxylaseTETActivity/InhibitionAssayKit(Fluorometric).

Fig.3. DemonstrationofhighsensitivityandspecificityoftheTETactivityassayachievedbyusingnuclearextractswiththetheEpigenase5-mCHydroxylaseTETActivity/InhibitionAssayKit(Fluorometric).NuclearextractswerepreparedfrommouseEScellsbyusingtheEpiQuikNuclearExtractionKit(Cat.No.OP-0002).

Fig.4. Illustratedstandardcurvegeneratedwiththekit"sTETassaystandard.