Isolation of Ribosomes by Chromatography

Abstract

Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.

Dissociation of Ribosomes into Large and Small Subunits

“…Resuspend the 70S ribosomal pellet from Protocol: Purification of 70S Ribosomes (Rivera et al 2014b) or the ribosomal pellet from Protocol: Isolation of Ribosomes by Chromatography (Maguire 2014) in a small volume of prokaryotic ribosome dissociation buffer. Measure the absorbance of the sample at OD 260 , and dilute the sample to 100-150 OD 260 units/mL with prokaryotic ribosome dissociation buffer.…”