N-Myristoyl Transferase (NMT)-Catalyzed Labeling of Bacterial Proteins for Imaging in Fixed and Live Cells

Methods for selective protein imaging are critical for elucidating how cells orchestrate fundamental biological processes. We recently developed a chemoenzymatic method to modify bacterial proteins in situ for fluorescence imaging using N-myristoyl transferase (NMT). Target proteins outfitted with an N-terminal NMT recognition sequence are covalently modified with an azido fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition allows for conjugation to cell-permeant fluorophores and imaging by fluorescence microscopy. Here we describe sample preparation and labeling protocols for imaging bacterial proteins in fixed and live cells.