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A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation
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Biology
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A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

DOI:
10.3791/4083-v

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14:44 min

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September 24, 2012

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, , , , ,
1Max Planck Institute of Molecular Plant Physiology, 2University of Kaiserslautern

Chapters

  • 00:05Title
  • 02:30Cell Lysis, Crosslinking and Sample Preparation
  • 05:22Immunoprecipitation
  • 07:06Sample Preparation for nano-LC-MS/MS
  • 10:50Results: HSP70B with its Nucleotide Exchange Factor CGE1
  • 14:07Conclusion

Summary

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We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Tags

Protein-protein Interactions15N Metabolic LabelingImmunoprecipitationQuantitative Mass SpectrometryAffinity ModulationCo-immunoprecipitationTargeted ApproachMass Spectrometry (LC-MS/MS)False Positive DiscoveriesRNAiQUICK (QUantitative Immunoprecipitation Combined With Knockdown)

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