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Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level
Published on: April 19, 2019
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Quantification of absolute labeling efficiency at the single-protein level.
Joschka Hellmeier1, Sebastian Strauss1, Shuhan Xu1
1Max Planck Institute of Biochemistry, Planegg, Germany.
Nature Methods
|April 24, 2024
Summary
Researchers developed a new method to accurately quantify protein labeling efficiency in super-resolution microscopy. This technique uses a reference tag and DNA-barcoded imaging to precisely measure single-protein binding.
Area of Science:
- Biophysics
- Molecular Biology
- Microscopy
Background:
- Super-resolution microscopy enables visualization of single proteins in cellular structures.
- Accurate quantification of protein organization and stoichiometry is limited by the lack of methods to evaluate absolute binder labeling efficiency.
Purpose of the Study:
- To introduce a universally applicable approach for evaluating absolute binder labeling efficiency at the single-protein level.
- To enable precise quantification of protein labeling in super-resolution imaging.
Main Methods:
- A reference tag was fused to the target protein of interest.
- High-affinity binders (antibodies or nanobodies) were attached to both the reference tag and the target protein.
- DNA-barcoded sequential super-resolution imaging was employed to correlate reference tag location with the target molecule binder.
Main Results:
- The developed approach allows for precise quantification of labeling efficiency at the single-protein level.
- This method overcomes the limitations of current techniques for evaluating binder labeling efficiency.
Conclusions:
- The introduced method provides a general solution for accurate binder labeling efficiency evaluation in super-resolution microscopy.
- This facilitates more precise analysis of protein organization and stoichiometry in biological systems.
