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Counting Proteins in Single Cells with Addressable Droplet Microarrays
Published on: July 6, 2018
Analysis of protein-protein interactions by using droplet-based microfluidics
Monpichar Srisa-Art1, Dong-Ku Kang, Jongin Hong
1Department of Chemistry, Imperial College London, Exhibition Road, London, UK.
Chembiochem : a European Journal of Chemical Biology
|June 5, 2009
Summary
Researchers validated a new method for measuring protein interactions using tiny droplets. This high-throughput platform accurately determines binding affinities, like those for angiogenin (ANG), without affecting protein activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Accurate quantification of protein-protein interactions is crucial for understanding biological processes.
- Traditional methods for measuring binding affinities can be time-consuming and require large sample volumes.
Purpose of the Study:
- To develop and validate a high-throughput screening method for analyzing protein-protein interactions.
- To assess the reliability of using picoliter-sized droplets for measuring dissociation constants (K(D)) of protein interactions.
- To confirm that fluorophore labeling does not interfere with protein function or antibody binding.
Main Methods:
- Utilized Förster Resonance Energy Transfer (FRET) analysis within thousands of picoliter-sized droplets.
- Performed bulk-fluorescence polarization measurements for comparative analysis.
- Assessed the impact of fluorophores on angiogenin (ANG) activity and antibody binding.
Main Results:
- K(D) values obtained from FRET analysis in picoliter droplets showed strong agreement with bulk-fluorescence polarization data.
- Fluorophore conjugation did not alter the biological activity of angiogenin (ANG).
- Antibody binding to ANG remained unaffected by the presence of fluorophores.
Conclusions:
- FRET analysis in picoliter droplets provides a robust and accurate method for determining protein-protein interaction K(D) values.
- This experimental approach is suitable for high-throughput screening of molecular interactions.
- The platform offers a sensitive and reliable alternative to traditional binding assays.
