Experimental workflow for ribosome separation and reconstitution (RSR). Ribosomes for a cell-free translation reaction can be isolated from a previously prepared CFE (A), or by the direct lysis of yeast cells (B). (a) CFE is ultracentrifuged at 180,000g (180K) for 2 h at 4°C, producing ribosome-containing pellet P180 and ribosome-free supernatant S180 (b). Ribosomal pellet P180 is solubilized (c) and added back to S180 (d), along with the energy regeneration system, amino acids, and (optionally) a reporter mRNA (e). Translation reactions are carried out at 21°C for 60–90 min (f). Alternatively, yeast cells are lysed with glass beads, and the clarified cellular lysate is next layered onto a 20% glycerol cushion (g); ribosomes are precipitated by centrifugation through the cushion at 180,000g (180K) for 2 h at 4°C (h). The resulting supernatant is discarded, ribosomal pellet P180 is solubilized (i) and added to the CFE-derived S180 to assemble the translation reaction (j).
