Figure 4.

Different strategies of genetic tagging. Protein of interest (POI, brown) can be fused with various tags to ensure fluorescence labeling in live cells. (A) Classical labeling with GFP-like fluorescent proteins (FP). Chromophore maturation in FP is a relatively slow process (tens of minutes), thus early events of POI trafficking and interactions are missed. This problem can be solved by immediate labeling of newly synthesized POI with tags depicted in B–D. (B) KECs labeling. A heterodimerizing pair of coils (positively charged K-coil and negatively charged E-coil) is used to attract preformed mature FP to nascent POI. It can be applied to POIs with distinct intracellular localization (fibers, membranes, etc.) to distinguish between target signal and diffuse FP background. (C) Labeling with bilirubin-binding fluorescent protein UnaG. (D) Labeling with fluorogen-activating proteins (FAP), which bind exogenously added fluorogenic dyes.