JournalofVisualizedExperimentswww.jove.comCopyright©2011CreativeCommonsAttribution-NonCommercialLicenseJuly2011|53|e3061|Page1of7VideoArticleVisualizationofRecombinantDNAandProteinComplexesUsingAtomicForceMicroscopyPatrickJ.M.Murphy1,MorganShannon2,JohnGoertz21CollegeofNursing,InterdisciplinaryLifeSciencesResearchLaboratory,SeattleUniversity2CollegeofScienceandEngineering,InterdisciplinaryLifeSciencesResearchLaboratory,SeattleUniversityCorrespondenceto:PatrickJ.M.Murphyatmurphyp@seattleu.eduURL:http://www.jove.com/video/3061DOI:doi:10.3791/3061Keywords:Bioengineering,Issue53,atomicforcemicroscopy,glucocorticoidreceptor,protein-proteininteraction,DNA-proteininteraction,scanningprobemicroscopy,immunoadsorptionDatePublished:7/18/2011Citation:Murphy,P.J.M.,Shannon,M.,Goertz,J.VisualizationofRecombinantDNAandProteinComplexesUsingAtomicForceMicroscopy.J.Vis.Exp.(53),e3061,doi:10.3791/3061(2011).AbstractAtomicforcemicroscopy(AFM)allowsforthevisualizingofindividualproteins,DNAmolecules,protein-proteincomplexes,andDNA-proteincomplexes.Ontheendofthemicroscope'scantileverisanano-scaleprobe,whichtraversesimageareasrangingfromnanometerstomicrometers,measuringtheelevationofmacromoleculesrestingonthesubstratesurfaceatanygivenpoint.Electrostaticforcescauseproteins,lipids,andnucleicacidstolooselyattachtothesubstrateinrandomorientationsandpermitimaging.Thegenerateddataresembleatopographicalmap,wherethemacromoleculesresolveasthree-dimensionalparticlesofdiscretesizes(Figure1)1,2.TappingmodeAFMinvolvestherepeatedoscillationofthecantilever,whichpermitsimagingofrelativelysoftbiomaterialssuchasDNAandproteins.OneofthenotablebenefitsofAFMoverothernanoscalemicroscopytechniquesisitsrelativeadaptabilitytovisualizeindividualproteinsandmacromolecularcomplexesinaqueousbuffers,includingnear-physiologicbufferedconditions,inreal-time,andwithoutstainingorcoatingthesampletobeimaged.ThemethodpresentedheredescribestheimagingofDNAandanimmunoadsorbedtranscriptionfactor(i.e.theglucocorticoidreceptor,GR)...
