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dc.contributor.authorLoveland, Anna B.
dc.contributor.authorHabuchi, Satoshi
dc.contributor.authorWalter, Johannes C.
dc.contributor.authorvan Oijen, Antoine M.
dc.date.accessioned2014-08-27T09:49:27Z
dc.date.available2014-08-27T09:49:27Z
dc.date.issued2012-09-09
dc.identifier.citationLoveland AB, Habuchi S, Walter JC, van Oijen AM (2012) A general approach to break the concentration barrier in single-molecule imaging. Nature Methods 9: 987-992. doi:10.1038/nmeth.2174.
dc.identifier.issn15487091
dc.identifier.pmid22961247
dc.identifier.doi10.1038/nmeth.2174
dc.identifier.urihttp://hdl.handle.net/10754/325368
dc.description.abstractSingle-molecule fluorescence imaging is often incompatible with physiological protein concentrations, as fluorescence background overwhelms an individual molecule's signal. We solve this problem with a new imaging approach called PhADE (PhotoActivation, Diffusion and Excitation). A protein of interest is fused to a photoactivatable protein (mKikGR) and introduced to its surface-immobilized substrate. After photoactivation of mKikGR near the surface, rapid diffusion of the unbound mKikGR fusion out of the detection volume eliminates background fluorescence, whereupon the bound molecules are imaged. We labeled the eukaryotic DNA replication protein flap endonuclease 1 with mKikGR and added it to replication-competent Xenopus laevis egg extracts. PhADE imaging of high concentrations of the fusion construct revealed its dynamics and micrometer-scale movements on individual, replicating DNA molecules. Because PhADE imaging is in principle compatible with any photoactivatable fluorophore, it should have broad applicability in revealing single-molecule dynamics and stoichiometry of macromolecular protein complexes at previously inaccessible fluorophore concentrations. © 2012 Nature America, Inc. All rights reserved.
dc.language.isoen
dc.publisherSpringer Nature
dc.rightsUsers may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms
dc.rightsArchived with thanks to Nature Methods
dc.subjectdeoxyribonuclease I
dc.subjectXenopus protein
dc.subjectconcentration (parameters)
dc.subjectdiffusion
dc.subjectDNA replication
dc.subjectmolecular imaging
dc.subjectphotoactivation
dc.subjectprotein analysis
dc.subjectprotein binding
dc.subjectprotein immobilization
dc.subjectXenopus laevis
dc.subjectDiffusion
dc.subjectDNA Replication
dc.subjectFlap Endonucleases
dc.subjectLuminescent Proteins
dc.subjectMicroscopy, Fluorescence
dc.subjectProliferating Cell Nuclear Antigen
dc.subjectEukaryota
dc.subjectXenopus laevis
dc.titleA general approach to break the concentration barrier in single-molecule imaging
dc.typeArticle
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Division
dc.identifier.journalNature Methods
dc.identifier.pmcidPMC3610324
dc.eprint.versionPost-print
dc.contributor.institutionDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States
dc.contributor.institutionGraduate Program in Biophysics, Harvard University, Cambridge, MA, United States
dc.contributor.institutionDepartment of Biochemistry, Brandeis University, Waltham, MA, United States
dc.contributor.institutionZernike Institute for Advanced Materials, University of Groningen, Groningen, Netherlands
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)
kaust.personHabuchi, Satoshi
refterms.dateFOA2018-06-13T15:22:28Z


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