ANALYTICALBIOCHEMISTRYAnalyticalBiochemistry364(2007)60–66www.elsevier.com/locate/yabio0003-2697/$-seefrontmatter©2007ElsevierInc.Allrightsreserved.doi:10.1016/j.ab.2007.01.040IdentiWcationoftissue-speciWcDNA–proteinbindingsitesbymeansoftwo-dimensionalelectrophoreticmobilityshiftassaydisplayIgorP.Chernov,KiraA.Timchenko,SergeyB.Akopov,LevG.Nikolaev¤,EugeneD.SverdlovShemyakin–OvchinnikovInstituteofBioorganicChemistry,RussianAcademyofSciences,117997Moscow,RussiaReceived11December2006Availableonline2February2007AbstractWedevelopedatechniqueofdiVerentialelectrophoreticmobilityshiftassay(EMSA)displayallowingidentiWcationoftissue-speciWcprotein-bindingsiteswithinlonggenomicsequences.Usingthisapproach,weidentiWed10celltype-speciWcprotein-bindingsites(proteintargetsites[PTSs])withina137-kbhumanchromosome19region.Ingeneral,tissue-speciWcbindingofproteinsfromdiVerentnuclearextractsbyindividualPTSsdidnotfollowtheall-or-nothingprinciple.Mostoften,PTS–proteincomplexeswereformedinallcases,buttheywerediVerentfordiVerentnuclearextractsused.©2007ElsevierInc.Allrightsreserved.Keywords:DNA–proteininteractions;Electrophoreticmobilityshiftassay(EMSA);DiVerentialdisplayThepublicationofthedrafthumangenomesequence[1,2]andsequencesofothermetazoangenomes[3]enabledpositioningandanalysisofvariousgenomefunc-tionalelements,Wrstofallcodingsequences[4,5].How-ever,completefunctionalannotationofsequencedeukaryoticgenomescannotbelimitedtoRNAcodingsequencesandshouldalsoincludepositioningofallnon-codingregulatoryelements.Unfortunately,experimentaldataongenomicpositionsofthemultitudeofregulatorysequences,suchasenhancers,promoters,transcriptionterminators,andreplicationorigins,areverylimited,especiallyatthewholegenomelevel.Ingeneral,mostgenomicregulatoryelements(e.g.,enhancers)aregenespeciWcandthereforehavediVerentstructures,inparticu-larbecausegenesorgroupofgenesusuallyareregulatedindependently.Therefore,predictionoftheseelementsbycomputationalmethodsisdiYcultandnotalwaysreliable.Mostofthegen...
