ChIPresultsobtainedwiththeantibodydirectedagainstH3K27me3S28p
ChIPassayswereperformedusinghumanHeLacellstreatedwithcolcemid,theantibodyagainstH3K27me3S28p(Cat.No.25245)andoptimizedPCRprimersetsforqPCR.ChIPwasperformedusingshearedchromatinfrom1millioncells.Atitrationoftheantibodyconsistingof1,2,5,and10μgperChIPexperimentwasanalysed.IgG(2μg/IP)wasusedasnegativeIPcontrol.QPCRwasperformedwithprimersforthepromoteroftheactivegeneGAPDHasanegativecontrol,andforthecodingregionsoftheinactivegenesMYT1andTSH2Baspositivecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).TheseresultsareinaccordancewiththeobservationthatH3K27me3S28pispreferablypresentatinactivegenes.

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3K27me3S28p
ChIPwasperformedonshearedchromatinfrom1.5millioncolcemidtreatedHeLaS3cellsusing2μgoftheantibodyagainstH3K27me3S28p(Cat.No.25245)asdescribedabove.TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaHiSeq.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer’sinstructions.The51bptagswerealignedtothehumangenomeusingtheBWAalgorithm.Figure2showstheenrichmentingenomicregionssurroundingtheTSH2BandMYT1positivecontrolgenes(fig2AandB,indicatedbyanarrow),andofchromosome3and20(figure2CandD).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodydirectedagainstH3K27me3S28p(Cat.No.25245).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure2),thetiteroftheantibodywasestimatedtobe1:4,300.

CrossreactivitytestoftheantibodydirectedagainstH3K27me3S28p
ADotBlotanalysiswasperformedtotestthecrossreactivityoftheantibodyagainstH3K27me3S28p(Cat.No.25245)withpeptidescontainingothermodificationsofhistoneH3andH4andtheunmodifiedH3K27S28sequence.Onehundredto0.2pmolofthepeptidecontainingtherespectivehistonemodificationwerespottedonamembrane.Theantibodywasusedatadilutionof1:2,000.Figure3showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3K27me3S28p
A.Westernblotwasperformedonhistoneextracts(15μg,lane1)fromHeLacells,andon1μgofrecombinanthistoneH3(lane2)usingtheantibodyagainstH3K27me3S28p(Cat.No.25245)diluted1:100inTBS-Tweencontaining5%skimmedmilk.Thepositionoftheproteinofinterestisindicatedontheright;theMarker(inkDa)isshownontheleft.
B.CellcycleexperimentusingtheantibodyagainstH3K27me3S28p.WBwasperformedoncellsblockedindifferentstagesofthecellcycle(1=asynchronouspopulation,2=G1/Sa,3=G1/Sb,4=Sphase,5=G2/M,6=M,7=G1).ThepictureshowsanicepeakofH3K27me3S28pexpressionduringmitosis.

Storeat–80°Cforupto2years.Centrifugeafterfirstthawtomaximizeproductrecovery.Aliquottoavoidrepeatedfreeze/thawcycles.Aliquotsmaybestoredat–20°Cforatleastonemonth.