After completion, cell-free reactions were quenched with formic acid to denature the proteins and stop the reactions. The reactions, in 384-well plates, were centrifuged at 3500g for 15 min to pellet any precipitated protein. For the acyl-CoA capture reactions, 3 μl of this cell-free reaction was transferred to a new 384-well plate, and the following species were added, bringing the final reaction volume to 8 μl with the final concentrations as follows: 100 mM phosphate buffer at pH 6.5, 40 mM EDTA, 0.9 mM capture peptide, and 0.1 mM normalization peptide. The well plates were sealed, and the reaction mixtures were incubated at 42°C for 3 hours. It is important to choose and appropriate concentration of capture peptide. If the concentration is too high, the signal from unreacted sensor peptide may overwhelm the signal from any captured species. In this work, the total added peptide concentration was chosen to be 1 mM across all reactions, a reasonable concentration relative to the expected yield from the best cell-free reactions while also giving good dynamic range for detection of acyl-species under low-yield conditions.

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