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Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics
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Biology
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Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

DOI:
10.3791/51656-v

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12:53 min

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July 06, 2014

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1Division of Virology, Department of Pathology,University of Cambridge

Chapters

  • 00:05Title
  • 01:49Harvesting of SILAC-labeled Cell Lysates
  • 03:39Binding Lysate to Anti-GFP Beads
  • 04:45Preparation of Samples for MS Analysis
  • 06:11Data Analysis I: Understanding the Results and Removal of Low-confidence Identifications
  • 07:36Data Analysis II: Selecting High Confidence Interactions for Further Study
  • 08:58Data Analysis III: Merging Replica Datasets
  • 10:59Results: Identification of Translation Initiation Factor – Binding Proteins
  • 12:22Conclusion

Summary

Automatic Translation

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SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Tags

Keywords: SILACImmunoprecipitationQuantitative ProteomicsProtein-protein InteractionsEIF4AIEIF4AIIMass SpectrometryData AnalysisStable Isotope LabelingAffinity Purification

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