Isolation of Preribosomes from HeLa Cells and Their Characterization by Electrophoresis on Uniform and Exponential‐Gradient‐Polyacrylamide Gels

Abstract

New methods are presented for the isolation of nucleoli and the extraction of intact preribosomes under mild conditions avoiding EDTA and high salt buffers. These nucleolar preribosomes were characterized by an improved technique of polyacrylamide‐gel electrophoresis which gives better resolution than sedimentation analysis for both ribonucleoprotein particles and free‐RNA molecules. Thus, the 50‐S native‐ribosomal subunit could be separated from its 55‐S precursor containing 32‐S pre‐RNA, and another nucleolar precursor, a “70‐S” particle, could be identified. The separation technique newly developed is disc electrophoresis using exponential polyacrylamide gels. The resolution with these gels is greater than that with uniform gels over a wider range of molecular weights. The final resolution is independent of the volume in which the sample is loaded and the diffusion of molecules during migration is eliminated. Molecular weights of RNA molecules can be estimated in exponential gels as in uniform gels. Electrophoresis in quartz tubes allows the direct scanning of ultraviolet absorbance during and after the run. As all handling is thus avoided, gels of very low concentration can be used.