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An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics
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Biochemistry
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JoVE Journal Biochemistry
An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

DOI:
10.3791/61648-v

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09:52 min

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September 15, 2020

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, ,
1Molecular Biology Program,Memorial Sloan Kettering Cancer Center

Chapters

  • 00:05Introduction
  • 00:51Equipment Preparation
  • 03:113’-End Biotinylated mRNA Immobilization
  • 05:20Translation Mix Assembly and Delivery
  • 07:24Results: Analyses for Saccharomyces cerevisiae Translation of a Firefly Luciferase-encoding mRNA
  • 09:12Conclusion

Summary

Automatic Translation

Automatic Translation

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Tags

Keywords: Single-molecule ImagingCap-dependent TranslationIn Vitro TranslationCell-free TranslationMicroscope SetupLaser ControlCamera SettingsSyringe PumpWash Protocol

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