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Quantification of absolute labeling efficiency at the single-protein level.

Joschka HellmeierSebastian StraussShuhan XuLuciano A MasulloEduard M UnterauerRafal KowalewskiRalf Jungmann
Published in: Nature methods (2024)
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.
Keyphrases
  • protein protein
  • high resolution
  • amino acid
  • binding protein
  • high throughput
  • photodynamic therapy
  • optical coherence tomography
  • single cell
  • high speed
  • circulating tumor
  • label free
  • nucleic acid