Related Experiment Video
Updated: Jun 17, 2026
11:11
Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction
Published on: August 15, 2013
Use of flow cytometric methods to quantify protein-protein interactions
Levi L Blazer1, David L Roman, Molly R Muxlow
1Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan, USA.
Current Protocols in Cytometry
|January 14, 2010
Summary
This study introduces a novel flow cytometry protein interaction assay (FCPIA) for quantifying protein binding affinities. The method immobilizes proteins on beads and uses fluorescence to measure interactions, enabling high-throughput screening.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Understanding protein-protein interactions is crucial for deciphering cellular mechanisms.
- Existing methods for quantifying these interactions can be complex or lack throughput.
- The regulator of G-protein signaling protein, RGS19, serves as a model for studying binding partner affinities.
Purpose of the Study:
- To describe a quantitative method for analyzing protein-protein interactions using flow cytometry.
- To enable the calculation of protein-protein interaction affinities.
- To adapt the assay for high-throughput screening applications.
Main Methods:
- Immobilizing proteins onto polystyrene beads.
- Incubating beads with fluorescently labeled binding partners.
- Quantifying bead-associated fluorescence using flow cytometry.
Main Results:
- The flow cytometry protein interaction assay (FCPIA) allows for quantitative analysis of protein binding.
- The method can determine interaction affinities in saturation or competition formats.
- An adaptation of the assay is suitable for high-throughput screening.
Conclusions:
- FCPIA provides a versatile and quantitative approach for studying protein-protein interactions.
- This assay facilitates the characterization of binding affinities and competition dynamics.
- The high-throughput compatibility expands its utility in drug discovery and biological research.
More Related Videos
Related Concept Videos
02:26Protein Networks
An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
01:23Flow Cytometry
The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
In...
In...
02:04Protein-protein Interfaces
Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide...
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...