Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group
Although various methods for selective protein tagging have been established, their ap plications are limited by the low fluorescent tagging efficiency of specific terminal regions of the native proteins of interest (NPIs). In this study, the highly sensitive fluorescence imaging of single NPIs was...
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| Veröffentlicht in: | International journal of molecular sciences 2022-05, Vol.23 (10), p.5841 |
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| description | Although various methods for selective protein tagging have been established, their ap plications are limited by the low fluorescent tagging efficiency of specific terminal regions of the native proteins of interest (NPIs). In this study, the highly sensitive fluorescence imaging of single NPIs was demonstrated using a eukaryotic translation mechanism involving a free carboxyl group of a cell-permeable fluorescent dye. In living cells, the carboxyl group of cell-permeable fluorescent dyes reacted with the lysine residues of acceptor peptides (AP or AVI-Tag). Genetically encoded recognition demonstrated that the efficiency of fluorescence labeling was nearly 100%. Nickel-nitrilotriacetic acid (Ni-NTA) beads bound efficiently to a single NPI for detection in a cell without purification. Our labeling approach satisfied the necessary conditions for measuring fluorescently labeled NPI using universal carboxyl fluorescent dyes. This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology. |
| doi_str_mv | 10.3390/ijms23105841 |
| format | Article |
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This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology.</description><identifier>ISSN: 1422-0067</identifier><identifier>ISSN: 1661-6596</identifier><identifier>EISSN: 1422-0067</identifier><identifier>DOI: 10.3390/ijms23105841</identifier><identifier>PMID: 35628651</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Bone cancer ; Carboxyl group ; Chromatography ; Dihydrofolate reductase ; Dyes ; E coli ; Efficiency ; Fluorescence ; Fluorescent dyes ; Fluorescent indicators ; Genetic code ; Labeling ; Lysine ; Marking ; Mitochondria ; Nitrilotriacetic acid ; Peptides ; Permeability ; Proteins ; Proteomics ; Signal to noise ratio</subject><ispartof>International journal of molecular sciences, 2022-05, Vol.23 (10), p.5841</ispartof><rights>2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). 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This approach is expected to be useful for resolving complex biological/ecological issues and robust single-molecule analyses of dynamic processes, in addition to applications in ultra-sensitive NPIs detection using nanotechnology.</description><subject>Bone cancer</subject><subject>Carboxyl group</subject><subject>Chromatography</subject><subject>Dihydrofolate reductase</subject><subject>Dyes</subject><subject>E coli</subject><subject>Efficiency</subject><subject>Fluorescence</subject><subject>Fluorescent dyes</subject><subject>Fluorescent indicators</subject><subject>Genetic code</subject><subject>Labeling</subject><subject>Lysine</subject><subject>Marking</subject><subject>Mitochondria</subject><subject>Nitrilotriacetic acid</subject><subject>Peptides</subject><subject>Permeability</subject><subject>Proteins</subject><subject>Proteomics</subject><subject>Signal to noise ratio</subject><issn>1422-0067</issn><issn>1661-6596</issn><issn>1422-0067</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNpdkc1LHTEUxUNp8avuui4BNy4czfdMNoI81ApWXdR1SDKZZx6ZyWsy48d_bwatPLu43Avnx-EeDgA_MDqmVKITv-ozoRjxhuEvYAczQiqERP11494GuzmvECKUcLkFtikXpBEc74Cn2_XorQ7wIkwxuWzdYB286vXSD0sYO3ijR__o4F2Ko_NDhvd5FvQGP86icfDJjw9FWLgQqt-uN0kPrrpzqXfaBAcXOpn4_BLgZYrT-jv41umQ3f773gP3F-d_Fr-q69vLq8XZdWUZJmOlOW-s5G1NW9F0xGCBtMG2q4nRVrCuMbQuJJKslVwaKrg1zGCju5YZ0TC6B07ffNeT6V07v5t0UOvke51eVNRefVYG_6CW8VFJzKgktBgcvhuk-HdyeVS9L6lDKOnilBURNS7T8LqgB_-hqzilocSbKYSlFBIV6uiNsinmnFz38QxGam5UbTZa8J-bAT7gfxXSVy8EnjI</recordid><startdate>20220523</startdate><enddate>20220523</enddate><creator>Kim, Jung Min</creator><creator>Kang, Young-Mi</creator><general>MDPI AG</general><general>MDPI</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-0409-7500</orcidid></search><sort><creationdate>20220523</creationdate><title>Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group</title><author>Kim, Jung Min ; 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In this study, the highly sensitive fluorescence imaging of single NPIs was demonstrated using a eukaryotic translation mechanism involving a free carboxyl group of a cell-permeable fluorescent dye. In living cells, the carboxyl group of cell-permeable fluorescent dyes reacted with the lysine residues of acceptor peptides (AP or AVI-Tag). Genetically encoded recognition demonstrated that the efficiency of fluorescence labeling was nearly 100%. Nickel-nitrilotriacetic acid (Ni-NTA) beads bound efficiently to a single NPI for detection in a cell without purification. Our labeling approach satisfied the necessary conditions for measuring fluorescently labeled NPI using universal carboxyl fluorescent dyes. 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| subjects | Bone cancer Carboxyl group Chromatography Dihydrofolate reductase Dyes E coli Efficiency Fluorescence Fluorescent dyes Fluorescent indicators Genetic code Labeling Lysine Marking Mitochondria Nitrilotriacetic acid Peptides Permeability Proteins Proteomics Signal to noise ratio |
| title | Optical Fluorescence Imaging of Native Proteins Using a Fluorescent Probe with a Cell-Membrane-Permeable Carboxyl Group |
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