Free cytosolic polysomes and membrane-bound polysomes were fractionated by density centrifugation as described previously (47 (link)). Briefly, cells were homogenized in cold sucrose buffer (0.25 M sucrose, 10 mM Tris–HCl, pH7.4, 1 mM magnesium acetate and protease inhibitors). The homogenates were centrifuged at 800g for 10 min at 4 °C and the supernatants were layered on 5 to 50% sucrose gradients prepared in a buffer containing 10 mM Tris–HCl, pH7.4 and 1 mM magnesium acetate. The gradients were centrifuged at 39,000 rpm in a Beckman SW41 rotor for 2.5 h at 4 °C.
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