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Journal Article

Quantification of absolute labeling efficiency at the single-protein level

MPS-Authors
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Hellmeier,  Joschka
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Strauss,  Sebastian
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Xu,  Shuhan
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;
IMPRS-ML: Martinsried, Max Planck Institute of Biochemistry, Max Planck Society;

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Masullo,  Luciano A.
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Unterauer,  Eduard M.
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;
IMPRS-ML: Martinsried, Max Planck Institute of Biochemistry, Max Planck Society;

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Kowalewski,  Rafal
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;
IMPRS-ML: Martinsried, Max Planck Institute of Biochemistry, Max Planck Society;

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Jungmann,  Ralf
Jungmann, Ralf / Molecular Imaging and Bionanotechnology, Max Planck Institute of Biochemistry, Max Planck Society;

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Citation

Hellmeier, J., Strauss, S., Xu, S., Masullo, L. A., Unterauer, E. M., Kowalewski, R., et al. (2024). Quantification of absolute labeling efficiency at the single-protein level. Nature Methods. doi:10.1038/s41592-024-02242-5.


Cite as: https://hdl.handle.net/21.11116/0000-000F-42C7-6
Abstract
State-of-the-art super-resolution microscopy allows researchers to spatially resolve single proteins in dense clusters. However, accurate quantification of protein organization and stoichiometries requires a general method to evaluate absolute binder labeling efficiency, which is currently unavailable. Here we introduce a universally applicable approach that uses a reference tag fused to a target protein of interest. By attaching high-affinity binders, such as antibodies or nanobodies, to both the reference tag and the target protein, and then employing DNA-barcoded sequential super-resolution imaging, we can correlate the location of the reference tag with the target molecule binder. This approach facilitates the precise quantification of labeling efficiency at the single-protein level.
Super-resolution imaging of reference and target structures enables precise determination of the labeling efficiency of high-affinity binding proteins in cells for improved quantitative assessment of protein organization at the single-molecule level.