Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study
1Department of Pharmacology, University of Illinois at Chicago, USA.
Journal of Visualized Experiments : Jove
|August 28, 2013
Summary
Bimolecular Fluorescence Complementation (BiFC) combined with flow cytometry offers a rapid and accurate method to study protein-protein interactions. This approach efficiently maps interaction regions, aiding in the discovery of regulatory factors.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein-protein interactions are crucial for cellular functions.
- Bimolecular Fluorescence Complementation (BiFC) is a sensitive method to visualize these interactions in living cells.
- Microscopy-based BiFC quantification can be limited by subjectivity and time.
Purpose of the Study:
- To develop and validate a high-throughput flow cytometry-based BiFC assay for quantifying protein-protein interactions.
- To apply this method for mapping interaction domains between AKAP-Lbc and PDE4D3.
Main Methods:
- Utilized Bimolecular Fluorescence Complementation (BiFC) with Venus fluorescent protein fragments (VN and VC).
- Fused VN and VC fragments to AKAP-Lbc and PDE4D3 proteins.
- Employed flow cytometry to quantify BiFC fluorescence signals in a large cell population.
Main Results:
- Successfully established a flow cytometry-based BiFC assay for high-throughput analysis.
- Quantified protein-protein interaction strength between AKAP-Lbc and PDE4D3 with high accuracy.
- Mapped specific regions within PDE4D3 essential for AKAP-Lbc interaction.
Conclusions:
- Flow cytometry-coupled BiFC provides a robust, efficient, and quantitative method for studying protein-protein interactions.
- This methodology facilitates the screening of interaction partners and regulatory factors.
- Enables precise mapping of protein interaction domains.
