Notes&TipsCharacterizationofheparin–livingbacteriainteractionsbychemiluminescenceelectrophoreticmobilityshiftassayJonghoonKang1,MyungSoogLee,DavidG.Gorenstein*SealyCenterforStructuralBiologyandDepartmentofBiochemistryandMolecularBiology,UniversityofTexasMedicalBranch,Galveston,TX77555,USAReceived18December2006Availableonline2February2007Theelectrophoreticmobilityshiftassay(EMSA)2isoneofthemostsensitivemethodsforstudyingDNA–proteininteractions.Chemiluminescence(CL)hasbeenusedasanalternativetoradioisotopicdetectionofsamplesintheEMSA[1,2]becauseithasadvantagessuchassafetyandstability(noisotopicdecay)ofthesample.Inthecurrentstudy,weexaminedthefeasibilityoftheapplicationofCLEMSAtostudyingheparin–livingbacteriainteractions.Asanexample,bindingofbiotinylatedheparintoEsche-richiacoliwasexamined.Thepathogenesisofmostinfectionsisinitiatedbymicrobialadhesiontohosttissue.Therefore,thisadhesionisapromisingtargetforthedevelopmentofnewantimicro-bialtherapeutics[3].Intheadhesion,heparinorheparin-relatedoligosaccharidesareoneoftheextracellularmatrixmoleculesofthehostrecognizedbycellsurfaceproteinsofbacteria[4–8].Duetothelackofappropriatetechniquesforthestudyofheparin–livingbacteriainteractions,mostheparin–bac-teriabindingstudieshavebeenconductedwithisolatedbacterialproteins[9,10].Althoughusefulinformationcanbeobtainedfromsuchstudies,onepotentialdrawbackistheexclusionofmembranephenomenasuchasligand-inducedreceptoroligomerizationthatcanaffecttheoverallbindingaffinity[11].Therefore,itisdesirabletoexaminetheadhesionpro-cessusingbacteriaintheintactstatetoincludethemem-branephenomena.Withthispurpose,scintillationcountingofradioisotoperadiation[12]andsodiumdodecylsulfate–polyacrylamidegelelectrophoresis[8]wereusedtoassessthebindingofheparintolivingbacteria.Morerecently,atomicforcemicroscopywasusedsuccessfullytoinvestigateheparin–livingbacteriainteractionsandpro-ducequantitativedata[13].However,thesemethodsarequalitative[8]andrequireradioisotopelabeling[12],aninstrumentthatmightno...
