Analysis of ribosomes before and after 180K-centrifugation. (A) One-step ultracentrifugation generates stable ribosomes and ribosome-free lysate. Aliquots of complete CFE (CFE), separated S180 and P180 (CFE: S, P), and P180 isolated from cells with glass-bead lysis (Cells: P) were resolved on a denaturing agarose gel and analyzed by northern hybridization with indicated probes. Prior to transfer onto nylon membrane, the gel was stained with SYBR Gold. (B,C) Ribosomes precipitated by one-step ultracentrifugation exist as 80S monosomes. (B) Complete CFE and solubilized P180 were centrifuged through 15%–45% (w/v) sucrose gradients and fractionated with continuous absorbance measurement at 254 nm to visualize ribosomal peaks. RNA was extracted from individual fractions and analyzed by northern hybridization as described in A. (C) Sucrose gradient centrifugation analysis performed with the total cellular lysate obtained with glass-bead lysis and its resuspended P180.
