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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.

Author information

1
Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
2
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
3
Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
4
Bioanalytics, Institute for Clinical Chemistry, University Medical Center Göttingen, Göttingen, Germany.
5
3D Electron Cryo-Microscopy Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

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.

KEYWORDS:

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

PMID:
26633879
PMCID:
PMC4755751
DOI:
10.7554/eLife.11349
[Indexed for MEDLINE]
Free PMC Article

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