Polyribosome Analysis
Polyribosome Analysis - Related Videos
09:16
Eukaryotic Polyribosome Profile Analysis
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Cited by 51 •
2010
This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.
09:13
In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation
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Cited by 14 •
2014
This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.
08:49
Peering at Brain Polysomes with Atomic Force Microscopy
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Cited by 9 •
2016
While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.
15:04
A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity
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Cited by 46 •
2009
This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.
10:39
Automated Analysis of Intracellular Phenotypes of Salmonella Using ImageJ
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Cited by 3 •
2022
Salmonella invades and replicates inside intestinal epithelial cells both in Salmonella-specific vacuoles and free in the cytosol (hyper-replication). A high-throughput fluorescence microscopy-based protocol is described here to quantify the intracellular phenotypes of Salmonella by two complementary image analyses through ImageJ, reaching single-cell resolution and scoring.
FAQs
The location of an mRNA or ribosomal component within the gradient reflects its association with separated ribosome-containing material. Comparing these distributions reveals whether particular transcripts are represented differently among fractions under distinct biological conditions. This provides a biochemical readout of changes in translation-related activity rather than relying only on measurements of gene expression.
Sucrose-density-gradient centrifugation separates components of a cell lysate according to their behavior during sedimentation through the gradient. Material associated with polyribosomes can therefore be distinguished from other lysate components and collected in separate fractions. Researchers then examine those fractions to determine where specific mRNAs and ribosomal components are distributed.
Researchers compare the fraction distribution of selected mRNAs between biological conditions. A transcript that shifts among ribosome-associated fractions may show altered engagement with the translation machinery, providing evidence of changed translational efficiency. This comparison is useful when RNA-level changes alone do not indicate whether the corresponding transcripts are being used differently for protein production.
Analyzing both types of material connects transcript distribution with the ribosomal machinery responsible for translation. The combined pattern helps distinguish changes in messenger RNA association from broader changes in ribosomal composition across the gradient. This relationship is important for interpreting how altered RNA-ribosome interactions may affect functional protein production in cells.
A typical workflow begins by preparing a cell lysate that preserves the relevant messenger RNA and ribosomal associations. The lysate is separated by sucrose-density-gradient centrifugation, after which polyribosome-containing fractions are collected. Researchers analyze the mRNAs and ribosomal components in those fractions, then compare their distributions across samples or experimental conditions.
The method is useful when investigators need to compare translational activity across conditions such as development, cellular stress, disease, or treatment. By examining how mRNAs and ribosomal components distribute across fractions, they can identify transcripts with altered translational efficiency and evaluate whether biological changes extend from gene expression to protein production.
Fraction comparisons indicate whether changes in gene expression are accompanied by differences in association with multiple ribosomes. This helps researchers assess the translational consequences of a condition, rather than stopping at transcript abundance. In biology, that distinction supports studies of regulation in which the final outcome depends on how effectively available mRNAs contribute to protein synthesis.