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Updated: Jul 15, 2026
A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
Published on: December 1, 2018
One-step analysis of protein complexes in microliters of cell lysate using indirect immunolabeling & fluorescence
Oda Stoevesandt1, Roland Brock
1Interfaculty Institute for Cell Biology, Department of Molecular Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
Insights
This study introduces a rapid "mix-and-measure" method for analyzing endogenous protein-protein interactions using minimal cell lysate. This technique avoids protein overexpression, enabling efficient monitoring of cellular signaling networks and compound screening.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Analyzing endogenous protein-protein interactions is crucial for understanding cellular signaling.
- Existing methods often require protein overexpression or extensive sample preparation, limiting their applicability.
Purpose of the Study:
- To develop a streamlined protocol for analyzing endogenous protein-protein interactions.
- To enable miniaturized and parallel analysis of protein interactions in crude cell lysates.
- To facilitate the screening of compounds that modulate protein interactions.
Main Methods:
- A 'mix-and-measure' procedure using indirect immunofluorescence labeling.
- Detection of interactions via fluorescence cross-correlation spectroscopy.
- Utilizes microliters of crude cell lysate without heterologous protein expression.
Main Results:
- Enables parallel and miniaturized analysis of protein-protein interactions.
- Avoids altering cellular processes through overexpression of tagged proteins.
- Suitable for monitoring signaling networks and screening interaction-interfering compounds.
Conclusions:
- The described protocol offers an efficient and sensitive method for studying endogenous protein interactions.
- This approach is valuable for dissecting signaling pathways and drug discovery.
- The method's minimal sample requirement and ease of use promote high-throughput analysis.
Abstract:
This protocol describes a 'mix-and-measure' procedure for the analysis of interactions of endogenous proteins in microliters of crude cell lysates. The proteins of interest are labeled by indirect immunofluorescence through simple addition of all primary and secondary antibodies to the lysate. Detection is based on fluorescence cross-correlation spectroscopy. Due to the minimal number of handling steps for sample preparation and the need of only microliters of sample, the approach enables the parallel and miniaturized analysis of protein-protein interactions. No heterologous expression of proteins with detection tags is required. For this reason, the cellular processes leading to protein-protein interactions are not skewed by overexpression of individual components. This makes the approach particularly suitable for the parallel monitoring of interactions in signaling networks. Additionally, the approach enables the screening and titration of compounds interfering with interactions, especially for those interactions based on signaling-dependent post-translational modifications. This protocol can be completed in approximately 22 h, including a 16-h incubation phase.
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