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General Laboratory Techniques

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Basic Methods in Cellular and Molecular Biology

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Model Organisms I

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 JoVE Biology

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

1Department of Biological Sciences, University of Alabama Huntsville, 2Department of Biology, Stanford University


JoVE 2498

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

 JoVE Biology

Chromatographic Purification of Highly Active Yeast Ribosomes

1Department of Cell Biology and Molecular Genetics, University of Maryland, 2Department of Biotechnology and Microbiology, Vilnius University


JoVE 3214

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

 JoVE Biology

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

1Center for Gene Regulation in Health and Disease, Department of Biological, Geological and Environmental Sciences, Cleveland State University


JoVE 4026

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

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