Abstract
This chapter describes the procedures for isolating active membrane-associated polysomes and free polysomes essentially free of each other; these procedures are applied successfully to both gram-positive and gram-negative bacteria to study the site of synthesis of certain proteins. Because most ribosomes are engaged in protein synthesis in exponentially growing cells, the gently prepared lysate of such cells is a rich source of polysomes. Membrane-associated polysomes are separated from free polysomes, based either on the lower density or on the larger size of the former. The fractionation based on differential density gave better separation. The most important considerations while isolating functional polysomes are preventing polysome runoff and preventing degradation by ribonuclease.
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