Covalent Protein Labeling by SpyTag-SpyCatcher in Fixed Cells for Super-Resolution Microscopy

Abstract

Labeling proteins with high specificity and efficiency is a fundamental prerequisite for microscopic visualization of subcellular protein structures and interactions. Although the comparatively small size of epitope tags makes them less perturbative to fusion proteins, they require the use of large antibodies that often limit probe accessibility and effective resolution. Here we use the covalent SpyTag-SpyCatcher system as an epitope-like tag for fluorescent labeling of intracellular proteins in fixed cells for both conventional and super-resolution microscopy. We also applied this method to endogenous proteins by gene editing, demonstrating its high labeling efficiency and capability for isoform-specific labeling.

Keywords: SpyTag-SpyCatcher; epitope tags; fluorescence microscopy; fluorescent probes; super-resolution microscopy.