ChIPresultsobtainedwiththeantibodydirectedagainstH3R17me2(asym)K18ac
ChIPassayswereperformedusinghumanHeLacells,theantibodyagainstH3R17me2(asym)K18ac(cat.No.25276)andoptimizedPCRprimerpairsforqPCR.ChIPwasperformedusingshearedchromatinfrom1,000,000cells.Atitrationconsistingof1,2,5and10µgofantibodyperChIPexperimentwasanalyzed.IgG(2µg/IP)wasusedasanegativeIPcontrol.QuantitativePCRwasperformedwithprimersforthepromotersoftheactiveEIF4A2andc-fosgenes,usedaspositivecontrolsandfortheinactiveMYOD1geneandtheSat2satelliterepeat,usedasnegativecontrols.Figure1showstherecovery,expressedasa%ofinput(therelativeamountofimmunoprecipitatedDNAcomparedtoinputDNAafterqPCRanalysis).

ChIP-seqresultsobtainedwiththeantibodydirectedagainstH3R17me2(asym)K18ac
ChIPwasperformedasdescribedaboveusing1µgoftheantibodyagainstH3R17me2(asym)K18ac(cat.No.25276).TheimmunoprecipitatedDNAwassubsequentlyanalysedonanIlluminaGenomeAnalyzer.Librarypreparation,clustergenerationandsequencingwereperformedaccordingtothemanufacturer"sinstructions.The36bptagswerealignedtothehumangenomeusingtheELANDalgorithm.Figure2showsthepeakdistributionalongthecompletehumanX-chromosomeandazoomintoa500kbregion(figure2AandB),andintworegionsonchromosome14and3surroundingthec-fosandEIF4A2positivecontrolgenes(figure2CandD,respectively).

Determinationoftheantibodytiter
Todeterminethetiteroftheantibody,anELISAwasperformedusingaserialdilutionoftheantibodyagainstH3R17me2(asym)K18ac(cat.No.25276).Theantigenusedwasapeptidecontainingthehistonemodificationofinterest.Byplottingtheabsorbanceagainsttheantibodydilution(Figure3),thetiteroftheantibodywasestimatedtobe1:28,800.

CrossreactivitytestsusingtheantibodydirectedagainstH3R17me2(asym)K18ac
TotestthecrossreactivityoftheantibodyagainstH3R17me2(asym)K18ac(cat.No.25276),aDotBlotanalysiswasperformedwithpeptidescontainingotherhistonemodificationsandtheunmodifiedH3R17K18.Onehundredto0.2pmoloftherespectivepeptideswerespottedonamembrane.Theantibodywasusedatadilutionof1:20,000.Figure4showsahighspecificityoftheantibodyforthemodificationofinterest.

WesternblotanalysisusingtheantibodydirectedagainstH3R17me2(asym)K18ac
Westernblotwasperformedonwholecell(25µg,lane1)andhistoneextracts(15µg,lane2)fromHeLacells,andon1µgofrecombinanthistoneH2A,H2B,H3andH4(lane3,4,5and6,respectively)usingtheantibodyagainstH3R17me2(asym)K18ac(cat.No.25276).Theantibodywasdiluted1:1,000inTBS-Tweencontaining5%skimmedmilk.TheMarker(inkDa)isshownontheleft.

ImmunofluorescenceusingtheantibodydirectedagainstH3R17me2(asym)K18ac
MouseNIH3T3cellswerestainedwiththeantibodyagainstH3R17me2(asym)K18ac(cat.No.25276)andwithDAPI.Cellswerefixedwith4%formaldehydefor10minutesandblockedwithPBS/TX-100containing5%normalgoatserumand1%BSA.ThecellswereimmunofluorescentlylabeledwiththeH3R17me2(asym)K18acantibody(left)diluted1:500inblockingsolutionfollowedbyananti-rabbitantibodyconjugatedtoAlexa488.ThemiddlepanelshowsstainingofthenucleiwithDAPI.Amergeofthetwostainingsisshownontheright. 

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