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One-step analysis of protein complexes in microliters of cell lysate using indirect immunolabeling & fluorescence cross-correlation spectroscopy

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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Figure 1: Overview of protein complex detection by indirect immunolabeling and FCCS.
Figure 2: Detection principle of FCCS.

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Acknowledgements

The authors thank N. Fischer for practical help with the experiments. R.B. gratefully acknowledges financial support from the Volkswagen Foundation ('Nachwuchsgruppen an Universitäten' I/77 472).

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Contributions

O.S. developed and validated the protocol, performed data analysis, and wrote the manuscript; R.B. developed the protocol and wrote the manuscript.

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Correspondence to Roland Brock.

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The authors declare no competing financial interests.

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Stoevesandt, O., Brock, R. One-step analysis of protein complexes in microliters of cell lysate using indirect immunolabeling & fluorescence cross-correlation spectroscopy. Nat Protoc 1, 223–229 (2006). https://doi.org/10.1038/nprot.2006.34

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  • DOI: https://doi.org/10.1038/nprot.2006.34

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