Host/IsotypeMouse/IgG1 Clone19C4B2.1 SpeciesReactivityAllspecies ValidationDataAc-KAntibodyWhitePaper | AAC03,anti-acetyllysineantibodyisapan-acetyllysinemousemonoclonalantibodythatispartoftheSignal-Seeker™productline.TheAnti-acetyl-lysineantibodyrecognizesproteinspost-translationallymodifiedbyacetylationontheepsilonaminegroupsoflysineresiduesthatoccuron30-50%ofallproteinsandinparticularhistones,p53,tubulinandmyosin.AproprietarymixtureofacetylatedproteinswasusedtoproduceahighlyrobustantibodythathasbeenshowntorecognizeawiderangeofacetylatedproteinsinIP,WB,ChIPandIFapplications.ThisAnti-acetyl-lysineantibodyhasmanyadvantageswhencomparedtoothercommerciallyavailableantibodiesasshownbelow.
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ValidatedApplications
WesternBlotusingAcetyl-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-LysineAntibody Fig2:Cos-7cellswereeithertreated(+)oruntreated(-)withTSA(1mM)andnicotinamide(1mM)for6hours.CelllysateswerepreparedinBlastRbufferandfiltersystemand1mgoflysateperreactionwasusedforIPofacetylatedproteins.20mlofAAC03wasusedperIPreaction.WesternblotsofimmunoprecipitatedproteinsweredevelopedusingAAC03-HRPat1:3000dilution. ToseethefullImmunoprecipitationcomparison,seetheOptimizedProtocolsortheproductdatasheet. |  |
ImmunofluorescenceusingAcetyl-LysineAntibody Fig3:Swiss3T3cells,untreated(aandc)ortreated(bandd)withTSA(1mMfor6h),werestainedasdescribedinthedatasheet.Acetylatedproteinswerevisualizedusingagreenfluorescentsecondary.ActinfiberswerevisualizedusingaredRhodaminePhalloidinandthenucleuswasstainedwithDAPI.TheacetylatedmicrotubulenetworkisclearlyvisIBLewithTSA-treatment,whilethegreenfluorescentnuclearintensityindicatethehighabundanceofacetylatedproteinsinthenucleus.Incandd,acetylatedBSA(10mg/ml)wasusedtocompeteforAAC03bindingasanindicatorofAAC02specificityforacetyl-lysinemodifications. ToseethefullImmunofluorescenceprotocol,seetheOptimizedProtocolsortheproductdatasheet. |  |
ChIPusingAcetyl-LysineAntibody Fig4:UtilizationofAAC03forChIP.ChromatinwaspreparedfromA431cells,eitheruntreatedortreatedwithTSA(1mM)andnicotinamide(1mM)for6hours.ChIPwasperformedasdescribed.mIgG:mouseIgGusedforChIPcontrol;AAC02:anti-acetyllysineantibodyusedforChIP;Input:celllysatepriortoChIP;H2O:WaterusedasPCRcontrol.ThePCRproductsobtainedwithGAPDHprimersare166bp. ToseetherecommendedChIPprotocol,seetheOptimizedProtocolsortheproductdatasheet. |  |
ProteinAcetylationBackground
Acetylationofproteinscanoccurasaco-translationalorpost-translationalmodification(PTM)(1).Co-translationalacetylationoccursattheN-terminalofapproximately85%ofmammalianproteins,itisirreversibleandisthoughttobeimportantinproteinstABIlity,localizationandsynthesis(1).Post-translationalacetylationoccursontheepsilonaminogroupoflysineresiduesasareversibleandhighlydynamicPTMthatisknowntobeakeyregulatorinmultiplecellularevents,includingchromatinstructure,transcription,metabolism,signaltransductionandcytoskeletalregulation(2-3).Todateover4,000proteinshavebeenidentifiedastargetsforPTMacetylationwhichiscomparabletophosphorylationincellularprevelance(3).AntibodyAAC01detectsacetyllysinePTMs.
References
1BogdanP.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,p67
Formoreinformationcontact:signalseeker@Cytoskeleton.com
AssociatedProducts:
Signal-Seeker™Acetyl-LysineDetectionKit(Cat.#BK163)
Signal-Seeker™:BlastR™RapidLysatePrepKit(Cat.#BLR01)
Signal-Seeker™AcetylationAffinityBeads(Cat.#AAC04-beads)
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