Journal
BIO-PROTOCOL
Volume 8, Issue 12, Pages -Publisher
BIO-PROTOCOL
DOI: 10.21769/BioProtoc.2881
Keywords
Cell-free protein synthesis; Selective double labeling; Unnatural amino acids; Precharged tRNA; Single-molecule FRET
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Funding
- 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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