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Identification of Protein Interacting Partners Using Tandem Affinity Purification
Published on: February 25, 2012
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Protein-Protein Interaction: Tandem Affinity Purification in Bacteria.
Julie P M Viala1, Emmanuelle Bouveret2
1Laboratoire d'Ingénierie des Systèmes Macromoléculaires, UMR7255, Institut de Microbiologie de la Méditerranée, Aix-Marseille University-CNRS, 31 Chemin Joseph Aiguier, 13009, Marseille, France. jviala@imm.cnrs.fr.
Methods in Molecular Biology (Clifton, N.J.)
|July 2, 2017
Summary
This study introduces tandem affinity purification (TAP) in bacteria to identify protein interaction partners under native conditions. This method helps understand cellular functions by revealing protein complexes and pathways.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Protein-protein interactions are crucial for cellular functions, forming complexes and pathways.
- Understanding these interactions deepens our knowledge of cell biology.
- Identifying protein partners is key to deciphering cellular machinery.
Purpose of the Study:
- To describe a protocol for tandem affinity purification (TAP) in bacteria.
- To enable the identification of protein interaction partners under native physiological conditions.
- To facilitate the study of protein complexes and cellular pathways.
Main Methods:
- Tandem Affinity Purification (TAP) using a dual-tag system (CBP and ProtA).
- Sequential purification steps utilizing IgG and calmodulin affinity resins.
- TEV protease cleavage to separate tagged protein and partners from contaminants.
- Chromosomal locus integration of the TAP-tag for native interaction studies.
Main Results:
- Successful identification of protein interaction partners under native conditions.
- Demonstration of TAP as an effective method for protein complex analysis in bacteria.
- Distinction between direct and indirect interaction partners.
Conclusions:
- Tandem affinity purification (TAP) is a robust method for identifying protein interaction networks in bacteria.
- This technique provides insights into protein complex formation and cellular pathway components.
- The protocol enables the study of protein interactions in a physiologically relevant context.
Keywords:
Affinity purificationCalmodulin binding peptide (CBP)Escherichia coliProtAProtein complexProtein–protein interactionSalmonellaTandem affinity purification (TAP)Tobacco etch virus (TEV)More Related Videos
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