Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate
Protein synthesis is by far the most energetically costly cellular process in rapidly dividing cells. Quantifying translating ribosomes in individual cells and their average mRNA transit rate is arduous. Quantitating assembled ribosomes in individual cells requires electron microscopy and does not i...
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Bio-protocol LLC
2025-01-01
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| author | Mina Seedhom Devin Dersh Jonathan Yewdell |
| author_facet | Mina Seedhom Devin Dersh Jonathan Yewdell |
| author_sort | Mina Seedhom |
| collection | DOAJ |
| description | Protein synthesis is by far the most energetically costly cellular process in rapidly dividing cells. Quantifying translating ribosomes in individual cells and their average mRNA transit rate is arduous. Quantitating assembled ribosomes in individual cells requires electron microscopy and does not indicate ribosome translation status. Measurement of average transit rates entails in vitro pulse-chase radiolabeling of isolated cells or ribosome profiling after ribosome runoff, which is expensive and extremely demanding technically. Here, we detail protocols based on ribosome-mediated nascent chain puromycylation, harringtonine to stall initiating ribosomes while allowing ribosome elongation to continue normally, and cycloheximide to freeze translating ribosomes in place. Each compound is delivered intravenously to mice in the appropriate order, and after ex vivo cell fixation and permeabilization, translating ribosome numbers and transit rates are measured by flow cytometry using a directly conjugated puromycin-specific antibody. |
| format | Article |
| id | doaj-art-e9d66db5457f4f12979d746885f9938c |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Bio-protocol LLC |
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| series | Bio-Protocol |
| spelling | doaj-art-e9d66db5457f4f12979d746885f9938c2025-02-07T08:16:38ZengBio-protocol LLCBio-Protocol2331-83252025-01-0115210.21769/BioProtoc.5165Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation RateMina Seedhom0Devin Dersh1Jonathan Yewdell2Diabetes Center of Excellence, Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USADepartment of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USANational Institute of Allergy and Infectious Diseases, Bethesda, MD, USAProtein synthesis is by far the most energetically costly cellular process in rapidly dividing cells. Quantifying translating ribosomes in individual cells and their average mRNA transit rate is arduous. Quantitating assembled ribosomes in individual cells requires electron microscopy and does not indicate ribosome translation status. Measurement of average transit rates entails in vitro pulse-chase radiolabeling of isolated cells or ribosome profiling after ribosome runoff, which is expensive and extremely demanding technically. Here, we detail protocols based on ribosome-mediated nascent chain puromycylation, harringtonine to stall initiating ribosomes while allowing ribosome elongation to continue normally, and cycloheximide to freeze translating ribosomes in place. Each compound is delivered intravenously to mice in the appropriate order, and after ex vivo cell fixation and permeabilization, translating ribosome numbers and transit rates are measured by flow cytometry using a directly conjugated puromycin-specific antibody.https://bio-protocol.org/en/bpdetail?id=5165&type=0 |
| spellingShingle | Mina Seedhom Devin Dersh Jonathan Yewdell Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate Bio-Protocol |
| title | Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate |
| title_full | Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate |
| title_fullStr | Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate |
| title_full_unstemmed | Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate |
| title_short | Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate |
| title_sort | flow based in vivo method to enumerate translating ribosomes and translation elongation rate |
| url | https://bio-protocol.org/en/bpdetail?id=5165&type=0 |
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