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Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation
Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with
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| Published in: | eLife 2015-12, Vol.4, p.e11349-e11349 |
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| creator | Pleiner, Tino Bates, Mark Trakhanov, Sergei Lee, Chung-Tien Schliep, Jan Erik Chug, Hema Böhning, Marc Stark, Holger Urlaub, Henning Görlich, Dirk |
| description | Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with |
| doi_str_mv | 10.7554/elife.11349 |
| format | article |
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We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.</description><identifier>ISSN: 2050-084X</identifier><identifier>EISSN: 2050-084X</identifier><identifier>DOI: 10.7554/elife.11349</identifier><identifier>PMID: 26633879</identifier><language>eng</language><publisher>England: eLife Science Publications, Ltd</publisher><subject>Antibodies ; Antigenic determinants ; Bacteria ; Biochemistry ; Cell Biology ; Comparative analysis ; Crosslinked polymers ; Cysteine ; Cystine ; epitope mapping ; Epitope Mapping - methods ; Fluorescence ; Fluorescence microscopy ; Humans ; label displacement ; Macromolecular Substances - immunology ; Macromolecular Substances - isolation & purification ; Mass spectrometry ; Nanobodies ; nanobody ; native purification ; nuclear pore complex ; Nuclear Pore Complex Proteins - immunology ; Nuclear Pore Complex Proteins - isolation & purification ; Optical Imaging - methods ; Protein binding ; Proteins ; Sensors ; Single-Domain Antibodies - metabolism ; site-specific ; Staining and Labeling - methods ; Tools and Resources ; Viral antibodies ; Wildlife conservation</subject><ispartof>eLife, 2015-12, Vol.4, p.e11349-e11349</ispartof><rights>COPYRIGHT 2015 eLife Science Publications, Ltd.</rights><rights>2015, Pleiner et al. This work is licensed under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/3.0/) (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015, Pleiner et al 2015 Pleiner et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5999-31500974f3acf42a6ee661225028a4051df9de57a9b0e4c4bc2a75f23ecadb1e3</citedby><cites>FETCH-LOGICAL-c5999-31500974f3acf42a6ee661225028a4051df9de57a9b0e4c4bc2a75f23ecadb1e3</cites><orcidid>0000-0002-5104-0315 ; 0000-0001-7103-5954 ; 0000-0003-1837-5233 ; 0000-0003-0668-5277 ; 0000-0002-1326-6153 ; 0000-0002-9889-7076 ; 0000-0002-4245-5113 ; 0000-0002-4343-5210</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/1953570877/fulltextPDF?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/1953570877?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,25753,27924,27925,37012,37013,44590,53791,53793,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26633879$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pleiner, Tino</creatorcontrib><creatorcontrib>Bates, Mark</creatorcontrib><creatorcontrib>Trakhanov, Sergei</creatorcontrib><creatorcontrib>Lee, Chung-Tien</creatorcontrib><creatorcontrib>Schliep, Jan Erik</creatorcontrib><creatorcontrib>Chug, Hema</creatorcontrib><creatorcontrib>Böhning, Marc</creatorcontrib><creatorcontrib>Stark, Holger</creatorcontrib><creatorcontrib>Urlaub, Henning</creatorcontrib><creatorcontrib>Görlich, Dirk</creatorcontrib><title>Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation</title><title>eLife</title><addtitle>Elife</addtitle><description>Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.</description><subject>Antibodies</subject><subject>Antigenic determinants</subject><subject>Bacteria</subject><subject>Biochemistry</subject><subject>Cell Biology</subject><subject>Comparative analysis</subject><subject>Crosslinked polymers</subject><subject>Cysteine</subject><subject>Cystine</subject><subject>epitope mapping</subject><subject>Epitope Mapping - methods</subject><subject>Fluorescence</subject><subject>Fluorescence microscopy</subject><subject>Humans</subject><subject>label displacement</subject><subject>Macromolecular Substances - immunology</subject><subject>Macromolecular Substances - isolation & purification</subject><subject>Mass spectrometry</subject><subject>Nanobodies</subject><subject>nanobody</subject><subject>native purification</subject><subject>nuclear pore complex</subject><subject>Nuclear Pore Complex Proteins - immunology</subject><subject>Nuclear Pore Complex Proteins - isolation & purification</subject><subject>Optical Imaging - methods</subject><subject>Protein binding</subject><subject>Proteins</subject><subject>Sensors</subject><subject>Single-Domain Antibodies - metabolism</subject><subject>site-specific</subject><subject>Staining and Labeling - methods</subject><subject>Tools and Resources</subject><subject>Viral antibodies</subject><subject>Wildlife conservation</subject><issn>2050-084X</issn><issn>2050-084X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNptks2P1CAUwBujcTfjnrybJl402hEolOLBZLPxY5KJJn4k3gilj8qmBRbazRr_eZmddd0xwgECv_cDHq8oHmO05ozRVzBaA2uMayruFccEMVShln6_f2d-VJykdI5y47RtsXhYHJGmqeuWi-Pi10flfOd7C-l1mewMVQqgrbG6HFWX7W4ojY9lWgLEKkLy4zJb70o7qSFvviyjCrYvIdjZB6gmFcIuRrm-dGq2l1CG6GewrtR-CiNclTY71M7xqHhg1Jjg5GZcFd_evf169qHafnq_OTvdVpoJIaoaM4QEp6ZW2lCiGoCmwYQwRFpFEcO9ET0wrkSHgGraaaI4M6QGrfoOQ70qNntv79W5DDFfPf6UXll5veDjIFWcrR5Bgmjarm0EJh2iyvQd4UAZQV1OJMVMZ9ebvSss3QS9BjdHNR5ID3ec_SEHfylp_i7OcBY8uxFEf7FAmuVkk4ZxVA78kiTmDRFNgzjK6NN_0HO_RJdTJbFgNeOo5fwvNaj8AOuMz-fqnVSeUl7TmtIMr4r1f6jce5is9g6MzesHAc8PAjIzw9U8qCUlufny-ZB9sWd19ClFMLf5wEjuylTCNpepvC7TTD-5m8Jb9k9R1r8BHsHjXw</recordid><startdate>20151203</startdate><enddate>20151203</enddate><creator>Pleiner, Tino</creator><creator>Bates, Mark</creator><creator>Trakhanov, Sergei</creator><creator>Lee, Chung-Tien</creator><creator>Schliep, Jan Erik</creator><creator>Chug, Hema</creator><creator>Böhning, Marc</creator><creator>Stark, Holger</creator><creator>Urlaub, Henning</creator><creator>Görlich, Dirk</creator><general>eLife Science Publications, Ltd</general><general>eLife Sciences Publications Ltd</general><general>eLife Sciences Publications, Ltd</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>88I</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</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><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-5104-0315</orcidid><orcidid>https://orcid.org/0000-0001-7103-5954</orcidid><orcidid>https://orcid.org/0000-0003-1837-5233</orcidid><orcidid>https://orcid.org/0000-0003-0668-5277</orcidid><orcidid>https://orcid.org/0000-0002-1326-6153</orcidid><orcidid>https://orcid.org/0000-0002-9889-7076</orcidid><orcidid>https://orcid.org/0000-0002-4245-5113</orcidid><orcidid>https://orcid.org/0000-0002-4343-5210</orcidid></search><sort><creationdate>20151203</creationdate><title>Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation</title><author>Pleiner, Tino ; Bates, Mark ; Trakhanov, Sergei ; Lee, Chung-Tien ; Schliep, Jan Erik ; Chug, Hema ; Böhning, Marc ; Stark, Holger ; Urlaub, Henning ; Görlich, Dirk</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5999-31500974f3acf42a6ee661225028a4051df9de57a9b0e4c4bc2a75f23ecadb1e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Antibodies</topic><topic>Antigenic determinants</topic><topic>Bacteria</topic><topic>Biochemistry</topic><topic>Cell Biology</topic><topic>Comparative analysis</topic><topic>Crosslinked polymers</topic><topic>Cysteine</topic><topic>Cystine</topic><topic>epitope mapping</topic><topic>Epitope Mapping - methods</topic><topic>Fluorescence</topic><topic>Fluorescence microscopy</topic><topic>Humans</topic><topic>label displacement</topic><topic>Macromolecular Substances - immunology</topic><topic>Macromolecular Substances - isolation & purification</topic><topic>Mass spectrometry</topic><topic>Nanobodies</topic><topic>nanobody</topic><topic>native purification</topic><topic>nuclear pore complex</topic><topic>Nuclear Pore Complex Proteins - immunology</topic><topic>Nuclear Pore Complex Proteins - isolation & purification</topic><topic>Optical Imaging - methods</topic><topic>Protein binding</topic><topic>Proteins</topic><topic>Sensors</topic><topic>Single-Domain Antibodies - metabolism</topic><topic>site-specific</topic><topic>Staining and Labeling - methods</topic><topic>Tools and Resources</topic><topic>Viral antibodies</topic><topic>Wildlife conservation</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pleiner, Tino</creatorcontrib><creatorcontrib>Bates, Mark</creatorcontrib><creatorcontrib>Trakhanov, Sergei</creatorcontrib><creatorcontrib>Lee, Chung-Tien</creatorcontrib><creatorcontrib>Schliep, Jan Erik</creatorcontrib><creatorcontrib>Chug, Hema</creatorcontrib><creatorcontrib>Böhning, Marc</creatorcontrib><creatorcontrib>Stark, Holger</creatorcontrib><creatorcontrib>Urlaub, Henning</creatorcontrib><creatorcontrib>Görlich, Dirk</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>AUTh Library subscriptions: ProQuest Central</collection><collection>ProQuest Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>ProQuest Science Journals</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>eLife</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pleiner, Tino</au><au>Bates, Mark</au><au>Trakhanov, Sergei</au><au>Lee, Chung-Tien</au><au>Schliep, Jan Erik</au><au>Chug, Hema</au><au>Böhning, Marc</au><au>Stark, Holger</au><au>Urlaub, Henning</au><au>Görlich, Dirk</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation</atitle><jtitle>eLife</jtitle><addtitle>Elife</addtitle><date>2015-12-03</date><risdate>2015</risdate><volume>4</volume><spage>e11349</spage><epage>e11349</epage><pages>e11349-e11349</pages><issn>2050-084X</issn><eissn>2050-084X</eissn><abstract>Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target.</abstract><cop>England</cop><pub>eLife Science Publications, Ltd</pub><pmid>26633879</pmid><doi>10.7554/elife.11349</doi><orcidid>https://orcid.org/0000-0002-5104-0315</orcidid><orcidid>https://orcid.org/0000-0001-7103-5954</orcidid><orcidid>https://orcid.org/0000-0003-1837-5233</orcidid><orcidid>https://orcid.org/0000-0003-0668-5277</orcidid><orcidid>https://orcid.org/0000-0002-1326-6153</orcidid><orcidid>https://orcid.org/0000-0002-9889-7076</orcidid><orcidid>https://orcid.org/0000-0002-4245-5113</orcidid><orcidid>https://orcid.org/0000-0002-4343-5210</orcidid><oa>free_for_read</oa></addata></record> |
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| source | Open Access: PubMed Central; Publicly Available Content Database |
| subjects | Antibodies Antigenic determinants Bacteria Biochemistry Cell Biology Comparative analysis Crosslinked polymers Cysteine Cystine epitope mapping Epitope Mapping - methods Fluorescence Fluorescence microscopy Humans label displacement Macromolecular Substances - immunology Macromolecular Substances - isolation & purification Mass spectrometry Nanobodies nanobody native purification nuclear pore complex Nuclear Pore Complex Proteins - immunology Nuclear Pore Complex Proteins - isolation & purification Optical Imaging - methods Protein binding Proteins Sensors Single-Domain Antibodies - metabolism site-specific Staining and Labeling - methods Tools and Resources Viral antibodies Wildlife conservation |
| title | Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation |
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