113.7K
views
59
authors
10
articles
Editors
2
Impact
Loading...
Review
02 November 2020
Nanobody-Based Probes for Subcellular Protein Identification and Visualization
Marit A. de Beer
 and 
Ben N. G. Giepmans

Understanding how building blocks of life contribute to physiology is greatly aided by protein identification and cellular localization. The two main labeling approaches developed over the past decades are labeling with antibodies such as immunoglobulin G (IgGs) or use of genetically encoded tags such as fluorescent proteins. However, IgGs are large proteins (150 kDa), which limits penetration depth and uncertainty of target position caused by up to ∼25 nm distance of the label created by the chosen targeting approach. Additionally, IgGs cannot be easily recombinantly modulated and engineered as part of fusion proteins because they consist of multiple independent translated chains. In the last decade single domain antigen binding proteins are being explored in bioscience as a tool in revealing molecular identity and localization to overcome limitations by IgGs. These nanobodies have several potential benefits over routine applications. Because of their small size (15 kDa), nanobodies better penetrate during labeling procedures and improve resolution. Moreover, nanobodies cDNA can easily be fused with other cDNA. Multidomain proteins can thus be easily engineered consisting of domains for targeting (nanobodies) and visualization by fluorescence microscopy (fluorescent proteins) or electron microscopy (based on certain enzymes). Additional modules for e.g., purification are also easily added. These nanobody-based probes can be applied in cells for live-cell endogenous protein detection or may be purified prior to use on molecules, cells or tissues. Here, we present the current state of nanobody-based probes and their implementation in microscopy, including pitfalls and potential future opportunities.

23,279 views
50 citations
Open for submission
Frontiers Logo

Frontiers in Cellular Neuroscience

Memory processing in health and disease: linking behavioral, circuits, and molecular scales.
Edited by Martín Cammarota, Fernando Castillo Díaz, Magdalena Pereyra
Deadline
10 August 2025
Submit a paper
Recommended Research Topics
Frontiers Logo

Frontiers in Cellular Neuroscience

Neural and Synaptic Defects in Autism Spectrum Disorders
Edited by Hansen Wang, Laurie Doering
280.4K
views
79
authors
24
articles
Frontiers Logo

Frontiers in Cellular Neuroscience

Development of the Striatum in Health and Disease
Edited by Marie-Christin Pauly, Màté Dániel Döbrössy
56.9K
views
30
authors
8
articles
Frontiers Logo

Frontiers in Cellular Neuroscience

Ion Channels: from Physiology to Channelopathies
Edited by Maria Cristina D'Adamo, Mauro Pessia
286.8K
views
172
authors
30
articles
Frontiers Logo

Frontiers in Cellular Neuroscience

Imaging the brain: advanced optical methods to investigate neural functions
Edited by Jonathan Mapelli, Egidio D‘Angelo
66.3K
views
45
authors
8
articles
Frontiers Logo

Frontiers in Molecular Neuroscience

Molecular Cell Biology of Cochlear Non-Sensory Cells: Roles in Hearing, Mechanisms of Hearing Loss, and Auditory Restoration
Edited by Hong-Bo Zhao, Fabio Mammano, Francisco Javier del Castillo
107.5K
views
120
authors
15
articles