TheEpiQuik™HistoneH3CitrullinationELISAKit (Colorimetric) isacompletesetofoptimizedbuffersandreagentsforspecificallymeasuringglobalhistoneH3citrullinationfromabroadrangeofspeciessuchasmammals,fungi,andbacteria,inavarietyofformsincludingculturedcellsandfreshtissues.Histoneextractscanbepreparedbyusingyourownsuccessfulmethod.Foryourconvenienceandthebestresults,Epigentekoffersahistoneextractionkitoptimizedforusewiththiskit.Histoneextractscanbeusedimmediatelyorstoredat–80°Cforfutureuse.Thiskithasthefollowingadvantages:

• Quickandefficientprocedure,whichcanbefinishedwithin3.5hours.
• Highsensitivityandspecificity.Thedetectionlimitisaslowas0.1ng/wellwithdynamicrangeof 0.5-10ng/wellwithintheindicatedamountrangeofthehistoneextracts.OnlyrecognizesH3citwithnocross-reactivitywithunmodifiedH3orothermodificationsatthesameargininesite.
• ThecontrolisconvenientlyincludedforthequantificationofH3cit.
• StripmicroplateformatmakestheassayflexIBLe:manualorhighthroughput.
• Simple,reliable,andconsistentassayconditions.

BackgroundInformation
Argininehistonecitrullinationisoneofthemanyimportantepigeneticmarks,andisessentialfortheregulationofepigeneticchromatinremodelingandimmunecell’sextracellulartrapprocesses.CitrullinatedarginineofhistoneH3(Arg2,8,17)regulatesgeneexpressionandismainlycatalyzedbypeptidylargininedeiminase4(PAD4).FurThermore,citrullinationofhistones,inparticularhistoneH3,wasrevealedasaconvergencepointfordiverseinflammatorysignalsthattriggertheneutrophilresponsetoinfections.ItwasreportedthatcitrullinatedhistoneH3couldbeapotentialserumbioMarkerfortheearlydiagnosisofsepticshock.MoreimmunologicaldiseasessuchasmultiplesclerosisandrheumatoidarthritisseemtobealsoassociatedwithchangeofhistoneH3citrullination.TheglobalhistoneH3citrullinationcanbechangedbyinhibitionoractivationofPADs.Therefore,quantitativedetectionofglobalhistoneH3citrullinationwouldprovideusefulinformationforbetterunderstandingepigeneticregulationofgeneactivationandsilencing,aswellasfordevelopingPAD-targeteddrugs. 

Principle&Procedure
ThiskitisdesignedformeasuringglobalH3cit.Inanassaywiththiskit,thehistoneproteinsarestablyspottedonthestripwells.ThecitrullineH3canberecognizedwithahigh-affinityantibodyanddetectedwithadetectionantibody,followedbyacolordevelopmentreagent.TheratioofH3citisproportionaltotheintensityofabsorbance.TheabsoluteamountofH3citcanbequantitatedbycomparingtothestandardcontrol.

StartingMaterials
Inputmaterialsshouldbehistoneextracts.Theamountofhistoneextractsforeachassaycanbe50ngto200ngwithanoptimalamountof100ng.

WB(10XWashBuffer)
BB(BlockingBuffer)
DS(DeveloperSolution)
SS(StopSolution)
HCB(HistoneCoatingBuffer)
8-WellAssayStrips(WithFrame)
ControlStrips(WithFrame)#
AdhesiveCoveringFilm
CAb(CaptureAntibody,1000X)*
DAb(DetectionAntibody,1000X)*
H3citControl(50µg/ml)

*Spinthesolutiondowntothebottompriortouse.
^#ControlStripsaregreentrimmedfordistinguishingfrom8-wellAssayStrips(forsamples). 

TheControlStripsareonlyforcontroluseandshouldnotbeusedforsampleassay.