Comparative analysis demonstrates that the ALFA-tag minimizes mRNA disruption and boosts fluorescence in living cells, indicating an improved method for imaging real-time translation.
Single-molecule imaging of translation sites in living cells has enabled the dynamics of protein synthesis to be investigated with high spatial and temporal resolution. These methodologies utilize the interaction between a multimerized epitope tag and its cognate fluorescent single-chain variable fragment or nanobody to detect nascent polypeptides as they emerge from the ribosome. Here, we present a systematic comparison of current methodologies and determine that the ALFA-tag reduces perturbations of mRNA expression and increases the fluorescent signal of translation sites.
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Misiaszek et al. (2026) studied this question.
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