1,2-Aminothiol-specific conjugation for dual-color fluorescent labeling via ultrafast TAMM conjugates

This chapter describes a practical protocol for dual-color fluorescent labeling of two different cell-surface proteins using ultrafast 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) conjugates. Each target protein is engineered with an N-terminal protease-recognition sequence that, upon cleavage by a specific protease, exposes an N-terminal cysteine and thus a 1,2-aminothiol handle for bioorthogonal TAMM conjugation. Accordingly, sequential protease treatment followed by labeling with distinct TAMM-fluorophore conjugates enables robust dual-color visualization of cell-surface proteins on mammalian cells.