生物
多克隆抗体
一级和二级抗体
荧光团
抗体
分子生物学
免疫染色
化学
融合蛋白
计算生物学
重组DNA
生物化学
荧光
免疫组织化学
免疫学
光学
物理
基因
作者
Tino Pleiner,Mark Bates,Dirk Görlich
标识
DOI:10.1083/jcb.201709115
摘要
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.
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