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Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions
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Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization.
Ting-Hai Xu1,2,3, Yan Yan1,2,3, Kaleeckal G Harikumar4
1Key Laboratory of Receptor Research, VARI-SIMM Center, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Bio-Protocol
|February 3, 2018
Summary
This study details a method to test protein-protein interactions using tagged proteins. It uses streptavidin beads to capture biotinylated and Flag-tagged proteins, assessing homooligomeric complex formation.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Protein-protein interactions are crucial for cellular functions.
- Conventional methods like pulldown assays are used to study these interactions *in vitro*.
- Assessing homooligomeric complex formation requires specific experimental designs.
Purpose of the Study:
- To present a protocol for determining protein-protein interactions *in vitro*.
- To demonstrate a method for assessing homooligomeric complex formation.
- To utilize tagged proteins for interaction studies.
Main Methods:
- Employing a pulldown assay technique.
- Expressing a protein of interest with two distinct tags.
- Utilizing streptavidin bead capture for biotinylated proteins.
- Co-precipitating Flag-tagged proteins using Streptavidin MagBeads.
Main Results:
- The protocol allows for the assessment of homooligomeric complex formation.
- Successful capture of biotinylated and co-associated Flag-tagged proteins was achieved.
- The method validates the interaction of tagged protein versions.
Conclusions:
- This protocol provides a reliable method for studying protein homooligomerization *in vitro*.
- The use of dual-tagged proteins and specific bead capture enhances interaction analysis.
- This technique is valuable for investigating protein complex formation in biochemical research.
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