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Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
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Immunology and Infection
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JoVE Journal Immunology and Infection
Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

DOI:
10.3791/51217-v

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11:33 min

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December 17, 2013

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, ,
1Genetics and Biochemistry Branch of the National Institute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health

Chapters

  • 00:05Title
  • 01:55Culture Preparation
  • 02:59Bpa Incorporation and Preparation for Photocrosslinking
  • 04:51Pulse-chase Labeling and UV Irradiation
  • 07:33Immunoprecipitation and Crosslinking Product Detection
  • 08:27Results: Interaction of the Autotransporter EspP wirh Molecular Chaperones and Components of the Bam Complex
  • 10:54Conclusion

Summary

Automatic Translation

Automatic Translation

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution “snapshots” of an ordered assembly pathway in a living cell.

Tags

Keywords: Site-specific CrosslinkingAmber SuppressionP-benzoylphenylalaninePhotocrosslinkingProtein-protein InteractionsBacterial Virulence FactorAssemblyPulse-chase LabelingImmunoprecipitationMass Spectrometry

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