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A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

Pleiner, Tino and Bates, Mark and Görlich, Dirk (2018) A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies. Journal of Cell Biology, 217 (3). pp. 1143-1154. ISSN 0021-9525. PMCID PMC5839796. http://resolver.caltech.edu/CaltechAUTHORS:20180404-110744971

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Abstract

Polyclonal anti–immunoglobulin G (anti-IgG) secondary antibodies are essential tools for many molecular biology techniques and diagnostic tests. Their animal-based production is, however, a major ethical problem. Here, we introduce a sustainable alternative, namely nanobodies against all mouse IgG subclasses and rabbit IgG. They can be produced at large scale in Escherichia coli and could thus make secondary antibody production in animals obsolete. Their recombinant nature allows fusion with affinity tags or reporter enzymes as well as efficient maleimide chemistry for fluorophore coupling. We demonstrate their superior performance in Western blotting, in both peroxidase- and fluorophore-linked form. Their site-specific labeling with multiple fluorophores creates bright imaging reagents for confocal and superresolution microscopy with much smaller label displacement than traditional secondary antibodies. They also enable simpler and faster immunostaining protocols, and allow multitarget localization with primary IgGs from the same species and of the same class.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://doi.org/10.1083/jcb.201709115DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839796/PubMed CentralArticle
https://doi.org/10.1101/209742DOIDiscussion Paper
ORCID:
AuthorORCID
Pleiner, Tino0000-0002-5104-0315
Bates, Mark0000-0003-0668-5277
Görlich, Dirk0000-0002-4343-5210
Alternate Title:A toolbox of anti-mouse and rabbit IgG secondary nanobodies
Additional Information:© 2018 Pleiner et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https ://creativecommons .org /licenses /by /4 .0 /). Submitted: 28 September 2017; Revision received 29 November 2017; Accepted: 7 December 2017; Published Online: 20 December, 2017. We thank Ulrike Teichmann and Rolf Rümenapf for alpaca care and immunization; Jens Krull, Heinz-Jürgen Dehne, Gabriele Hawlitscheck, Renate Rees, Jürgen Schünemann, and Tanja Gilat for excellent technical assistance; Philip Gunkel, Volker Cordes, and Mary Osborn for advice and antibodies (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany); Bastian B. Hülsmann for anti-Xenopus Nup nanobodies and antigens (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany); and Nataliia Naumenko and Trevor Huyton for critical reading of the manuscript. We thank Max-Planck-Gesellschaft and the Deutsche Forschungsgemeinschaft (SFB1190) for funding this work. T. Pleiner and D. Görlich are inventors on a European patent application encompassing the nanobodies described in this article. The authors declare no further conflict of interest. Author contributions: T. Pleiner: conception and design, acquisition of data, analysis and interpretation of data, drafting and revising the article; M. Bates: experiment design, acquisition of data, analysis and interpretation of data, drafting and revising the article; D. Görlich: conception and design, analysis and interpretation of data, drafting and revising the article.
Funders:
Funding AgencyGrant Number
Max-Planck-GesellschaftUNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)SFB1190
PubMed Central ID:PMC5839796
Record Number:CaltechAUTHORS:20180404-110744971
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20180404-110744971
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:85600
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:04 Apr 2018 18:25
Last Modified:26 Oct 2018 21:55

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