Abstract
This chapter discusses the methods for the isolation of a highly active, hydrodynamically uniform fraction of ribosomal couples from B. subtilis W168. Preincubation of extracts and high-salt washing are avoided because of the likelihood of degradation or depletion of ribosomal components during such procedures. Instead, ribosomes are fractionated and characterized by high-resolution sucrose density gradient centrifugation. The procedures described in this chapter are carried out under ionic conditions very similar to those found to be optimal for in vitro function of ribosomes. The final preparation of ribosomal couples are shown to be virtually devoid of endogenous mRNA activity and are highly dependent on the addition of a high-salt ribosomal wash fraction for activity. The ribosomes isolated as described here represent the most native material available for study of protein synthesis in gram-positive systems.
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