Core Concepts
Streptavidin-based imaging of TurboID-tagged proteins provides superior detection of proteins within phase-separated cellular regions and significantly boosts signal intensity compared to antibody-based methods, enabling improved visualization in applications like expansion microscopy and correlative light-electron microscopy.
Abstract
The content describes a comparative analysis of streptavidin-based and antibody-based protein visualization methods. Key highlights and insights:
Streptavidin imaging of TurboID-tagged proteins can accurately localize target proteins, with resolution comparable to antibody-based immunofluorescence in standard light microscopy.
Streptavidin can effectively detect target proteins within phase-separated cellular regions, such as the nuclear pore channel, nucleolus, and stress granules, where antibodies often fail to bind or penetrate. This is likely due to the dense, disordered nature of these phase-separated environments.
Streptavidin imaging provides a significantly stronger signal compared to antibody-based detection, with up to a 2.9-fold increase in maximum fluorescence intensity. This advantage is particularly beneficial for imaging applications that require reduced antigen density, such as expansion microscopy and correlative light-electron microscopy (CLEM).
Streptavidin-based imaging can provide additional insights into protein interactions and dynamics. The biotinylation by TurboID can reveal historic interactions and dynamic localization changes that are not captured by steady-state antibody labeling.
The authors systematically mapped the phase-separated regions of the trypanosome nuclear pore complex by comparing streptavidin and antibody accessibility, identifying the FG-repeat nucleoporins lining the central channel as inaccessible to antibodies.
In summary, the content demonstrates the superior performance of streptavidin-based imaging of TurboID-tagged proteins, particularly in visualizing proteins within phase-separated cellular compartments and enhancing imaging sensitivity in advanced microscopy techniques.
Stats
Streptavidin signal was 2.9-fold higher in maximum fluorescence intensity compared to anti-HA antibody for NUP158-TurboID-HA in trypanosome cells.
Quotes
"Streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, can provide information on protein interactions and biophysical environment."
"Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus or RNA granules, were readily detected with streptavidin, while most antibodies fail to label proteins in these environments."