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Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies

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BIO-PROTOCOL
卷 8, 期 12, 页码 -

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BIO-PROTOCOL
DOI: 10.21769/BioProtoc.2881

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Cell-free protein synthesis; Selective double labeling; Unnatural amino acids; Precharged tRNA; Single-molecule FRET

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  1. Forschungszentrum Julich

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Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET studies. The protocol describes two cell-free approaches to achieve a selective label scheme that allows the highest possible accuracy in inter-dye distance determination.

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