A toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons
Jie-Xian Dong,
Yongam Lee,
Michael Kirmiz,
Stephanie Palacio,
Camelia Dumitras,
Claudia M Moreno,
Richard Sando,
L Fernando Santana,
Thomas C Südhof,
Belvin Gong,
Karl D Murray,
James S Trimmer
Affiliations
Jie-Xian Dong
Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
Yongam Lee
Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
Michael Kirmiz
Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
Stephanie Palacio
Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
Camelia Dumitras
Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States
Claudia M Moreno
Department of Physiology and Membrane Biology, University of California, Davis, Davis, United States; Department of Physiology and Biophysics, University of Washington, Seattle, United States
Richard Sando
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford School of Medicine, Stanford, United States
Department of Neurobiology, Physiology and Behavior, University of California, Davis, Davis, United States; Department of Physiology and Membrane Biology, University of California, Davis, Davis, United States
Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.
