Quantifying Ribosome-Bound mRNA Levels

For ribosome association of individual mRNAs, gel electrophoresis following polysome analysis and fractionation was performed as described above. RNA was subsequently transferred to nitrocellulose membranes (AmershamHybond™–N, GE Healthcare) and further processed as described ^{31 (link)}. DNA oligonucleotides (Supplementary Information Table S2 [X85, X96, X105, X114]) were radiolabeled with PNK according to standard procedures. Probes were incubated with the membranes in hybridization buffer (6 × SSC, 0.1% SDS, 10 × Denhardt’s reagent) overnight at 42°C. Membranes were subsequently washed three times with wash buffer (6 × SSC, 0.1% SDS). Probe signals were visualized by a Molecular Dynamics Phosphorimager (GE Healthcare) and quantified by ImageQuant 5.2 software (Molecular Dynamics). Normalized signal intensities were compiled for fractions corresponding to monosomes, light and heavy polysomes and averaged from at least three biological replicates.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Guenther U.P., Weinberg D.E., Zubradt M.M., Tedeschi F.A., Stawicki B.N., Zagore L.L., Brar G.A., Licatalosi D.D., Bartel D.P., Weissman J.S, & Jankowsky E. (2018). The helicase Ded1p controls use of near-cognate translation initiation codons in 5′UTRs. Nature, 559(7712), 130-134.

Publication 2018

AmershamHybond™–N, Molecular Dynamics Phosphorimager ImageQuant 5.2 software

Biological Buffer Electrophoresis Fractionation Hybridization Light Membranes Molecular dynamics Monosomes Mrnas Nitrocellulose Oligonucleotides Polysomes Ribosome

Corresponding Organization :

Other organizations : Case Western Reserve University, University of California, San Francisco, University of California, Berkeley, Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute

Top 5 similar protocols

Variable analysis

independent variables

Ribosome association of individual mRNAs

dependent variables

Probe signals
Normalized signal intensities for fractions corresponding to monosomes, light and heavy polysomes

control variables

RNA transfer to nitrocellulose membranes
DNA oligonucleotide radiolabeling with PNK
Probe incubation with membranes in hybridization buffer
Membrane washing with wash buffer
Probe signal visualization and quantification

controls

No positive or negative controls explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!