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J Cell Biol. 2018 Mar 5;217(3):1143-1154. doi: 10.1083/jcb.201709115. Epub 2017 Dec 20.

A toolbox of anti-mouse and anti-rabbit IgG secondary nanobodies.

Author information

1
Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany tino.pleiner@mpibpc.mpg.de.
2
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany mark.bates@mpibpc.mpg.de.
3
Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany goerlich@mpibpc.mpg.de.

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.

PMID:
29263082
PMCID:
PMC5839796
DOI:
10.1083/jcb.201709115
[Indexed for MEDLINE]
Free PMC Article

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