Labeling of Phospho-Specific Antibodies with oYo-Link® Epitope Tags for Multiplex Immunostaining

Spatial proteomics has recently garnered significant interest, as it offers to provide unprecedented insight into biological processes in both health and disease, by connecting protein expression patterns from the subcellular level to the tissue or even organism level. These high-content approaches generally rely on a high degree of multiplexing, whereby multiple proteins can be detected simultaneously. The most versatile multiplexing approaches utilize antibodies to confer specificity for various intracellular proteins of interest. Therefore, these methods must be able to differentiate many antibodies at once. In this chapter, we describe a simple and rapid approach to labeling antibodies with distinct epitope tags in a site-specific manner. This allows multiple antibodies, even from the same host species, to be uniquely identified and detected and offers a simple approach for spatial proteomic applications.