Pulse-Chase in the Light Microscope

To visualize specific proteins in living cells, cell biologists have fused the green fluorescent protein (GFP) or its analogs to the C termini of proteins and expressed these chimeras in culture and in embryos (1–3). By using fluorescent proteins with different spectral properties (4–6), researchers can follow exogenous proteins in real time and map their cellular locations and interactions. These techniques have been complemented by the development of methods that permit the fluorescent labeling of recombinant proteins containing a tetracysteine sequence CCXXCC (where X is any amino acid). Proteins carrying this motif within an -helix can be stained in vivo with the nonfluorescent, membrane-permeant biarsenical derivative of fluorescein, FlAsH-EDT2 [4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2]. FlAsH-EDT2 binds with high affinity (Kd ~ 10 pM) to the tetracysteine motif, resulting in strong gree