Host/IsotypeMouse/IgG1Clone19C4B2.1SpeciesReactivityAllspeciesValidationDataAc-KAntibodyWhitePaperAAC03,anti-acetyllysineantibodyisapan-acetyllysinemousemonoclonalantibodythatispartoftheSignal-Seeker™productline.TheAnti-acetyl-lysineantibodyrecognizesproteinspost-translationallymodifiedbyacetylationontheepsilonaminegroupsoflysineresiduesthatoccuron30-50%ofallproteinsandinparticularhistones,p53,tubulinandmyosin.AproprietarymixtureofacetylatedproteinswasusedtoproduceahighlyrobustantibodythathasbeenshowntorecognizeawiderangeofacetylatedproteinsinIP,WB,ChIPandIFapplications.ThisAnti-acetyl-lysineantibodyhasmanyadvantageswhencomparedtoothercommerciallyavailableantibodiesasshownbelow.ValidatedApplicationsWesternBlotusingAcetyl-LysineAntibody(AAC03)Fig1:A:Murinetissueextract,30mgbrainextract.B:30mgofCos-7celllysatetreatedwithTSAandnicotinamide(+)oruntreated(-).Stronglyenhancedbandsat55and14-16kDainTSA-treatedlysatecorrespondtoacetylatedtubulinandhistoneproteins,respectively.C:TitrationofacetylatedBSA.Lanes1-5contain0.5,0.1,0.05,0.01,and0.005ngAc-BSA,lanes6-7contain500and1000ngnon-acetylatedBSA,respectively.AAC03wasusedata1:500dilutionfollowingtherecommendedwesternblotprotocol.ToseethefullWesternblotcomparison,seetheOptimizedProtocolsortheproductdatasheet.ImmunoprecipitationusingAcetyl-LysineAntibodyFig2:Cos-7cellswereeithertreated(+)oruntreated(-)withTSA(1mM)andnicotinamide(1mM)for6hours.CelllysateswerepreparedinBlastRbufferandfiltersystemand1mgoflysateperreactionwasusedforIPofacetylatedproteins.20mlofAAC03wasusedperIPreaction.WesternblotsofimmunoprecipitatedproteinsweredevelopedusingAAC03-HRPat1:3000dilution.ToseethefullImmunoprecipitationcomparison,seetheOptimizedProtocolsortheproductdatasheet.ImmunofluorescenceusingAcetyl-LysineAntibodyFig3:Swiss3T3cells,untreated(aandc)ortreated(bandd)withTSA(1mMfor6h),werestainedasdescribedinthedatasheet.Acetylatedproteinswerevisualizedusingagreenfluorescentsecondary.ActinfiberswerevisualizedusingaredRhodaminePhalloidinandthenucleuswasstainedwithDAPI.TheacetylatedmicrotubulenetworkisclearlyvisIBLewithTSA-treatment,whilethegreenfluorescentnuclearintensityindicatethehighabundanceofacetylatedproteinsinthenucleus.Incandd,acetylatedBSA(10mg/ml)wasusedtocompeteforAAC03bindingasanindicatorofAAC02specificityforacetyl-lysinemodifications.ToseethefullImmunofluorescenceprotocol,seetheOptimizedProtocolsortheproductdatasheet.ChIPusingAcetyl-LysineAntibodyFig4:UtilizationofAAC03forChIP.ChromatinwaspreparedfromA431cells,eitheruntreatedortreatedwithTSA(1mM)andnicotinamide(1mM)for6hours.ChIPwasperformedasdescribed.mIgG:mouseIgGusedforChIPcontrol;AAC02:anti-acetyllysineantibodyusedforChIP;Input:celllysatepriortoChIP;H2O:WaterusedasPCRcontrol.ThePCRproductsobtainedwithGAPDHprimersare166bp.ToseetherecommendedChIPprotocol,seetheOptimizedProtocolsortheproductdatasheet.ProteinAcetylationBackgroundAcetylationofproteinscanoccurasaco-translationalorpost-translationalmodification(PTM)(1).Co-translationalacetylationoccursattheN-terminalofapproximately85%ofmammalianproteins,itisirreversibleandisthoughttobeimportantinproteinstABIlity,localizationandsynthesis(1).Post-translationalacetylationoccursontheepsilonaminogroupoflysineresiduesasareversibleandhighlydynamicPTMthatisknowntobeakeyregulatorinmultiplecellularevents,includingchromatinstructure,transcription,metabolism,signaltransductionandcytoskeletalregulation(2-3).Todateover4,000proteinshavebeenidentifiedastargetsforPTMacetylationwhichiscomparabletophosphorylationincellularprevelance(3).AntibodyAAC01detectsacetyllysinePTMs.References1BogdanP.andShermanF.2002.Thediversityofacetylatedproteins.GenomeBiol.3(5):reviews0006.2LundbyA.etal.2012.Proteomicanalysisoflysineacetylationsitesinrattissuesrevealsorganspecificityandcellularpatterns.CellReports2:419-431.3SadoulK.etal.2010.Thetaleofproteinlysineacetylationinthecytoplasm.J.Biomed.Biotech.2011:1-15.4GolemisEAet.Al,Protein-ProteinInteractions,CSHLP,2005,p67Formoreinformationcontact:signalseeker@Cytoskeleton.comAssociatedProducts:Signal-Seeker™Acetyl-LysineDetectionKit(Cat.#BK163)Signal-Seeker™:BlastR™RapidLysatePrepKit(Cat.#BLR01)Signal-Seeker™AcetylationAffinityBeads(Cat.#AAC04-beads)