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

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Journal Bio Protoc
Specialty Biology
Date 2021 Jul 21
PMID 34285993
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Abstract

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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