OriginalarticlePrinciplesandproblemsoftheelectrophoreticmobilityshiftassayNeilS.Holdena,⁎,ClaireE.TaconbaDepartmentofCellBiologyandAnatomy,AirwayInflammationResearchGroup,FacultyofMedicine,UniversityofCalgary,Calgary,Alberta,CanadaT2N4N1bDepartmentofPhysiologyandPharmacology,AirwayInflammationResearchGroup,FacultyofMedicine,UniversityofCalgary,Calgary,Alberta,CanadaT2N4N1abstractarticleinfoArticlehistory:Received31December2009Accepted11March2010Keywords:EMSAMethodsSupershiftDNARadiolabelledEnd-labellingTroubleshooting32PAutoradiographyIntroduction:Theelectrophoreticmobilityshiftassay(EMSA)isclassicallyusedtodetectDNAbindingproteins,thetenetoftheEMSAisthatDNAwithproteinbound,migratesthroughapolyacrylamidegelmoreslowlythanthecorrespondingfreeunboundDNA.Methods:TheclassicalEMSAprotocolhas4majorsteps:1)Theisolationofproteinsfromcells.SincethevastmajorityofactiveDNAbindingproteinsarepresentwithinthenucleus,asequentialmembranelysisprotocolisusedwhichyieldspurifiednuclearprotein.2)ManufactureandradiolabellingoftheDNAprobe.Phosphorous32(32P)isattachedtothe5′endsoftheDNAprobethroughuseof32P-γATPasasubstrateforT4polynucleotidekinase.DNAprobescanbothbepurchasedorcustommade.3)PurifiedproteinsandradiolabelledDNAprobesareco-incubatedwithanEMSAbindingbuffertopromotebindingoftheproteinswiththeDNAprobe.IfasupershiftEMSAisbeingcarriedout,thereactionalsocontainsaselectiveantibodywhichwhenboundtotheprotein–DNAcomplexes,causesfurtherretardationwithinthegel.4)TheDNA–proteincomplexesareloadedandrunonanon-denaturingpolyacrylamidegelcausingseparationoftheDNA–proteincomplexesfromthefreeDNAprobes.Thepolyacrylamidegelsarethendrieddownandanalysedviaautoradiography.Results:Asademonstrationoftheeffectivenessofthisprotocol,weshowthattumournecrosisfactor(TNF)αandphorbol12-myristate13-acetate(PMA)stimulationofA549cells,resultsinanumberofDNA–proteincomplexesbeinginducedwhencomparedtountreatedcells.Wealsodemonstratethatthesecomplexescontainthep50andp65subunitsofNF-κBthroughutilisationofthe...
