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 \textless2 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.
%0 Journal Article
%1 Pleiner2015
%A Pleiner, Tino
%A Bates, Mark
%A Trakhanov, Sergei
%A Lee, Chung Tien
%A Schliep, Jan Erik
%A Chug, Hema
%A Böhning, Marc
%A Stark, Holger
%A Urlaub, Henning
%A Görlich, Dirk
%D 2015
%I eLife Sciences Publications Ltd
%J eLife
%K antibodies imaging microscopy nanobodies superresolution
%N DECEMBER2015
%R 10.7554/eLife.11349
%T Nanobodies: Site-specific labeling for super-resolution imaging, rapid epitope- mapping and native protein complex isolation
%V 4
%X 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 \textless2 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.
@article{Pleiner2015,
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 {\textless}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.},
added-at = {2020-04-06T13:11:22.000+0200},
author = {Pleiner, Tino and Bates, Mark and Trakhanov, Sergei and Lee, Chung Tien and Schliep, Jan Erik and Chug, Hema and B{\"{o}}hning, Marc and Stark, Holger and Urlaub, Henning and G{\"{o}}rlich, Dirk},
biburl = {https://www.bibsonomy.org/bibtex/2d5d1ade7fd1cdef09748d739897c1ce2/kfriedl},
doi = {10.7554/eLife.11349},
file = {:C$\backslash$:/Users/Karoline/AppData/Local/Mendeley Ltd./Mendeley Desktop/Downloaded/Pleiner et al. - 2015 - Nanobodies Site-specific labeling for super-resolution imaging, rapid epitope- mapping and native protein comple.pdf:pdf},
interhash = {779ed88a56e0850811ca8a8201d7f53f},
intrahash = {d5d1ade7fd1cdef09748d739897c1ce2},
issn = {2050084X},
journal = {eLife},
keywords = {antibodies imaging microscopy nanobodies superresolution},
month = dec,
number = {DECEMBER2015},
pmid = {26633879},
publisher = {eLife Sciences Publications Ltd},
timestamp = {2020-04-06T14:39:33.000+0200},
title = {{Nanobodies: Site-specific labeling for super-resolution imaging, rapid epitope- mapping and native protein complex isolation}},
volume = 4,
year = 2015
}