ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K9ac
ChIPwasperformedasdescribedaboveusing1μgoftheantibodyagainstH3K9ac(cat.No.25266).TheimmunoprecipitatedDNAwassubsequentlyanalysedwithanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletesequenceanda800kbregionoftheX-chromosome(figure2AandB)andin100kbregionssurroundingtheGAPDHandc-fosgenes(figure2CandD).TheseresultsclearlyshowanenrichmentofH3K9acatthepromotersofactivegenes.
ChIPresultsobtainedwiththeantibodydirectedagainstH3K9ac
ChIPassayswereperformedusingHeLacells,theantibodyagainstH3K9ac(cat.No.25266)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QuantitativePCRwasperformedwithprimersforthepromoteroftheactivec-fosandGAPDHgenes,usedaspositivecontrols,andforthecodingregionoftheinactiveMBgeneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K9acetylationisenrichedatthepromotersofactivegenes.
Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K9ac(cat.No.25266)inantigencoatedwells.Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:59,600.
CrossreactivitytestusingtheantibodydirectedagainstH3K9ac
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K9ac(cat.No.25266)withpeptidescontainingotherhistonemodificationsandtheunmodifiedH3K9sequence.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.ThisantibodyrecognizestheH3K9acetylation,bothinthepresenceandtheabsenceoftheK14acetylationortheS10phosphorylation.
WesternblotanalysisusingtheantibodydirectedagainstH3K9ac
HistoneextractsofHeLacells(15μg)wereanalysedbyWesternblotusingtheantibodyagainstH3K9ac(cat.No.25266)diluted1:1,000inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.
Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.
ELISA(1:1000)
DB(1:20,000)
WB(1:750)