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Free keywords:
SUPERRESOLUTION MICROSCOPY; EGF RECEPTOR; DIMERIZATION; ACTIVATION;
LIMITBiochemistry & Molecular Biology;
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.
