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Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling
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Biochemistry
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JoVE Journal Biochemistry
Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

DOI:
10.3791/60432-v

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11:19 min

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November 17, 2019

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, ,
1Department of Molecular, Cellular and Developmental Biology,University of Colorado, 2Linda Crnic Institute for Down Syndrome,University of Colorado School of Medicine

Chapters

  • 00:04Title
  • 01:07Preparation of Nuclear Extract
  • 03:45Immunoprecipitation of Endogenous Nuclear Bait Protein
  • 07:01Sample Preparation and LC/MS System Suitability
  • 09:09Results: A Single-bait Single-antibody IP-MS Experiment
  • 10:25Conclusion

Summary

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Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Tags

Keywords: Label-freeImmunoprecipitationMass SpectrometryNuclear InteractomeProtein-protein InteractionsSubcellular FractionationAffinity PurificationProteomics WorkflowCell LysisNuclear LysateDounce Homogenization

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