Biacore

WhatcanBiacoredoforyou?SPR(surfaceplasmonresonance)technologyenablesreal-timedetectionandmonitoringofbiomoleculareventsandprovidesquantitativeinformationon:1.Specificity–howspecificisthebindingbetweentwomoelcules?2.Concentration–howmuchofagivenmoleculeispresentandactive?3.Kinetics–whatistherateofassociation/dissociation?4.Affinity–howstrongisthebinding?5.Bindingpartners-provideidentificationofbindingtargetsbylinkingSPRtoMSThesensorgramprovidesquantitativeinformationinreal-timeonspecificityofbinding,activeconcentrationofmoleculeinasample,kineticsandaffinity.WhatisSPR?SPR(surfaceplasmonresonance)ariseswhenlightisreflectedundercertainconditionsfromaconductingfilmattheinterfacebetween2mediaofdifferentrefractiveindex.InBiacoresystems,themediaaresampleandglassofthesensorchip,andtheconductingfilmisathinlayerofgoldonthechipsurface.SPRcausesareductionintheintensityofreflectedlightataspecificangleofreflection.Thisanglevarieswiththerefractiveindexclosetothesurfaceonthesideoppositefromthereflectedlight(sampleside).SPRresponsevaluesareexpressedinresonanceunits(RU).OneRU=0.0001o.Formostproteinsthisisaboutachangeinconc.Of1pg/mm2onthesensorsurface.IllustrationofdetectorandIFCBiacoreterminologyligandanalyteligandanalytecapturingmolecule•Ligand:theinteractantimmobilizedonthesensorsurfaceTheligandcanbeimmobilizeddirectlytothesensorsurface,orindirectlyviaanimmobilizedcapturingmolecule•Analyte:theinteractantinfreesolutionSensorChipsQuantitativemeasurementsofthebindinginteractionbetweenoneormoremoleculesaredependentontheimmobilizationofatargetmoleculetothesensorchipsurface.Bindingpartnerstothetargetcanbecapturedfromacomplexmixture,inmostcases,withoutpriorpurification(forexample,clinicalmaterial,cellculturemedia)astheypassoverthechip.Interactionsbetweenproteins,nucleicacids,lipids,carbohydratesandevenwholecellscanbestudied.Thesensorchipconsistsofaglasssurface,coatedwithathinlayerofgold.Thisformsthebasisforarangeofspecializedsurfacesdesignedtooptimizethebindingofavarietyofmolecules.Inthemostwidelyusedsensorchip,thegoldsurfaceismodifiedwithacarboxymethylateddextranlayer.Thisdextranhydrogellayerformsahydrophilicenvironmentforattachedbiomolecules,preservingtheminanon-denaturedstate.ArangeofotherderivatizedsurfacesisalsoavailabletoenablevariousimmobilizationchemistriesBiacoreoffersarangeofSensorChipsSensorChipCM5–forapplicationsfrombasicresearchtoqualitycontrolSensorChipSA–forcaptureofbiotinylatedpeptides,proteinsandDNASensorChipNTA–forcaptureofligandsviametalchelationSensorChipHPA–formembranebiochemistryandthestudyofmembraneassociatedreceptorsinanearnativeenvironmentPioneerChips–astoolsforusers’novelapplicationsintermsofchemistryorbindingspecificity.SensorChipsCM5(carboxymethyldextran)Interactionsinvolvingalltypesofbiomoleculessuchasproteins,lipids,carbohydrates,andnucleicacidscanbestudied.Studiescanalsobemadeofreagentsranginginsizefromsmallorganicmolecules,e.g.drugcandidates,tolargemolecularassemblies,suchasmembranereceptorsembeddedinlipidbilayervesiclesorevenwholecells.AminecouplingAminecoupling:mostcommonmethodofcoupling.Surfaceisactivatedwitha1:1mixtureofNHS:EDC,togivereactivesucinimideesters.Theligand(inabuffergivinga+charge)ispassedoverthesurfaceandtheestersreactspontaneouslywithaminogroups.Anyfreeaminegroupcanreactwiththesurface.COHOCOOCNHOEDC/NHSNOONH2ligandligandAmineCouplingSensogramThiolcouplingThiolcoupling:anactivedisulfidemoietycanbeintroducedeitheronthedextranmatrixorontheligandmolecule.2-(2-pyrdinyldithio)ethaneamine(PDEA)canbeusedtointroducetheacticedi-sulfidegroup.COHOCOOCNHOEDC/NHSNOOSHligandligandPDEACNHOSSSSNSensorChipsSA(streptavidin)SensorChipSAisdesignedtobindbiotinylatedmolecules.Streptavidin-biotincouplingprovidesanalternativetootherligandcouplingmethodse.g.covalentamineorthiolcoupling,andisparticularlyvaluableforapplicationsinmolecularbiologywhereimmobilizationofnucleicacidsthrougha5'-biotinmoietyisoftenthemostpracticalapproach.SensorChipNTASensorChipNTAisdesignedtobindhistidine-taggedmolecules.Attachmentofligandsviahistidine-tagsprovidesavaluableandconvenientmethodologicalinterfacebetweenBIAanalysisandotherlaboratorytechniquesthatrelyonHis-tags,e.g.chelatingchromatography.AttachmentviaaHis-taghasalsotheadvantageoforientingtheligandmoleculesinahomogeneouswayandallowingtheattachmenttobecarriedoutwithoutsignificantlychangingthepHorionicstrengthduringthecouplingprocedure.B-cat/TCFactivationofcertainpromotersrequiressplicingvariantscontainingan“E-tail”UsingtheBiacoreweshowedthatTCF1E-tailbindsnon-specificallytoDNA(Syedetal2005)-5051015202530354004080120160200240280320360400Response(RU)Time(s)ka=1.03e+05/Mskd=2.49e-02/sKA=4.14e+06/MKD=2.42e-07Mss/dsDNAcompetionGST-Etail25nM024602004006008001000[ssMOP]nMRU(x10)TCF1E-tailspecificallybindsdsDNATCF1E-tailbindingtoDNAisduemainlytoelectrostaticinteractions