TheChromaFlash™ChromatinIsolation&ShearingKitisacompletesetofoptimizedbuffersandreagentsforisolatingchromatinorDNA-proteincomplexfrommammaliancellsortissuesinasimpleandrapidformat,followedbyashearingstepbysonication.Chromatinpreparedbythiskitcanbeusedinavarietyofchromatinimmunoprecipitationmethods.ItisalsotherecommendedmethodforobtainingchromatinrequiredbyEpigentek’sone-hourChIPmethodusingtheChromaFlash™One-StepChIPKit.Theisolatedchromatincanalsobeusedinotherchromatin-relatedapplicationssuchasinvitroprotein-DNAbindingassaysandnuclearenzymeassays. WhenperformingChIP,chromatinorDNA-proteincomplexincellsortissuesshouldfirstbeisolatedandshearedtoadesiredsize.TheChromaFlash™ChromatinIsolation&ShearingKitaddressestheinconvenienceandtimeconsumingissuesofexistingchromatinpreparationmethodsbyintroducingthefollowingfeatures: - Fastprocedure:theentireprocedurefromcell/tissuesampletoready-to-useshearedchromatinislessthan2hours.
- ConvenientandflexIBLe:thekitissuitableforpreparingbothnativechromatinandcross-linkedchromatinfrommonolayerorsUSPensioncells,orfromtissues.
- Chromatinshearingstepispre-optimizedforsonicationwithvarioussonicationplatformsincludingwaterbath-based(EpiSonic,COVARIS,Bioruptor)sonicators,probe-basedsonicators(Branson,Misonix), andenzyme-basedmethods.
BackgroundInformation
Chromatinimmunoprecipitaton(ChIP)offersanadvantageoustoolforstudyingprotein-DNAinteraction.TheexperimentercandetermineifaspecificproteinbindstothespecificsequencesofageneinlivingcellsbycombiningChIPwithPCR(ChIP-PCR),microarray(ChIP-chip),orsequencing(ChIP-Seq)techniques.Forexample,themeasurementoftheamountofmethylatedhistoneH3atlysine9(meH3-K9)associatedwithaspecificgenepromoterregionundervariousconditionscanbeachievedthroughaChIP-PCRassay,whilerecruitmentofmeH3-K9tothepromotersonagenome-widescalecanbedetectedbyChIP-chip.Inparticular,aChIPmethodwithspecificantibodiesdirectlyagainstvarioustranscriptionalfactorsiswidelydemanded. Principle&Procedure
TheChromaFlash™ChromatinIsolation&ShearingKitcontainsallreagentsrequiredforcarryingoutsuccessfulchromatinextractionandshearingdirectlyfrommammaliancellsortissues.Cellmembranesofthesample,withorwithoutcross-linking,arebrokendownusingtheprovidedlysisbuffer.ChromatinorDNA-proteincomplexisthenextractedwiththeextractionbuffer.Theextractedchromatinisthenshearedaccordingtotheprotocol"srecommendedprotocolsettingsforavarietyofsonicatorsoftheexperimenter"schoiceandstoredatanappropriatetemperatureafterbeingdilutedwithchromatinbuffer. StartingMaterials&InputAmount
Startingmaterialscanincludevarioustissueorcellsamplessuchascellsfromflaskormicroplateculturedcells,freshandfrozentissues,etc.Theamountofcellsandtissuesforeachpreparationcanbe2 x105 to 1x107 cellsand10mgto400mg,respectively.Foroptimalpreparation,theinputamountshouldbe2to5 x106 cellsor100to200mgtissues. Atotalof100standardextractions(using 2 x106 cellsor100mgoftissueperextraction)canbeperformedwiththiskit.Theyieldofshearedchromatinisapproximately3µgper 106 cellsorper50mgoftissue. | 
 Fig.1.SchematicprocedureoftheChromaFlash™ChromatinIsolation&ShearingKit.
Fig.2. ChromatinwasextractedfromsixMDA-231cellsamples.40ul(8ug)ofchromatinwereshearedina0.2mltubeusingtheEpiSonic1100for20cycles(15secOnand30secOff).DNAwaspurifiedandthenrunonagel.
Fig.3. TheshearedchromatinwasusedforChIP-qPCRanalysisofRNApolymeraseIIenrichmentinGAPDHandMLH1promotersbyusingtheChromaFlash™ One-StepChIPKit(P-2025)andtheMethylamp™MS-qPCRFastKit(P-1028).
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