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Related Concept Videos

Immunoprecipitation01:20

Immunoprecipitation

5.6K
Immunoprecipitation, or IP, is a widely used technique that employs protein-antibody interactions to isolate proteins or protein complexes in their native state for studying protein-protein interactions, quaternary structures, or supramolecular complexes. Various modifications of the technique, including chromatin IP, cross-linking IP, and fluorescence IP, are commonly used.
Chromatin Immunoprecipitation
Chromatin immunoprecipitation, also known as ChIP, is used to study protein-DNA or...
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Related Experiment Video

Updated: Aug 29, 2025

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography
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Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

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DSP-crosslinking and Immunoprecipitation to Isolate Weak Protein Complex.

Kotaro Akaki1, Takashi Mino1, Osamu Takeuchi1

  • 1Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.

Bio-Protocol
|September 9, 2022
PubMed
Summary

This study introduces a new method using dithiobis(succinimidyl propionate) crosslinking and tandem immunoprecipitation to detect weak protein-protein interactions. This technique enhances the identification of proteins interacting with your protein of interest.

Keywords:
Dithiobis(succinimidyl propionate) (DSP) crosslinkingFLAG-tagHeLa cellsHemagglutinin (HA)-tagImmunoprecipitation (IP)Protein-protein interaction (PPI)Tandem affinity purification

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Protein-protein interactions (PPIs) are crucial for understanding molecular regulation.
  • Standard immunoprecipitation methods can miss weakly interacting proteins.
  • Stabilizing PPIs is key to comprehensive detection.

Purpose of the Study:

  • To present a novel crosslinking method for improved PPI detection.
  • To enhance the identification of proteins interacting with proteins of interest (POIs).
  • To provide a robust technique applicable to various cytoplasmic proteins.

Main Methods:

  • Utilizing dithiobis(succinimidyl propionate) (DSP) for protein crosslinking.
  • Employing tandem immunoprecipitation with FLAG and HA tags.
  • Analyzing eluted proteins via mass spectrometry or western blotting.

Main Results:

  • DSP crosslinking stabilizes weak PPIs, preventing loss during analysis.
  • Tandem immunoprecipitation effectively enriches interacting proteins.
  • The method successfully identifies co-precipitated binding proteins.

Conclusions:

  • The DSP-mediated crosslinking followed by tandem immunoprecipitation is an effective strategy for detecting weak PPIs.
  • This technique improves the comprehensiveness of protein interaction studies.
  • The method is versatile and applicable to a wide range of cytoplasmic proteins.