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Elife. 2015 Dec 3;4:e11349. doi: 10.7554/eLife.11349.

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation.

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Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany.
3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.


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.


E. coli; biochemistry; cell biology; epitope mapping; label displacement; nanobody; native purification; nuclear pore complex; site-specific; xenopus

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