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The Use of Co-immunoprecipitation to Study Conformation-Specific Protein Interactions of Oligomeric α-Synuclein Aggregates

  • Cristine Betzer
  • Rikke Hahn Kofoed
  • Poul Henning Jensen
Protocol
Part of the Neuromethods book series (NM, volume 144)

Abstract

The development of aggregates of specific disease-associated proteins represents a common denominator for many neurodegenerative disorders. The gain of function of the aggregates is hypothesized to initiate pro-degenerative signaling pathways that cause neuronal dysfunctions and ultimately death of affected neurons. Comparing the protein interactome of the native normal functioning disease-associated protein to the interactome of the aggregated forms of the same protein may reveal disease-conducting signaling hubs of relevance to specific diseases. Here, we describe the experimental setup we used to identify specific interaction partners of soluble oligomeric α-synuclein aggregates including step-by-step protocols for preparation of antibody-conjugated Sepharose beads, purification of recombinant soluble α-synuclein oligomers, preparation of synaptosomal extracts from porcine brain, and the actual co-immunoprecipitation. Our goal is to present the reader issues for consideration before starting co-immunoprecipitation experiments and a practical overview of the technical finesses. This approach can be applied to study interaction of any purified disease-linked soluble aggregates.

Key words

Immunoprecipitation α-synuclein Oligomers Conformational-specific interactions Tissue fractionation Synaptosomes Tandem mass spectrometry Label-free quantification Skyline targeted proteomic environment 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Danish Research Institute of Translational Neuroscience—DANDRITEAarhus UniversityAarhusDenmark
  2. 2.Department of BiomedicineAarhus UniversityAarhusDenmark

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