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Journal Article

Selective cross-linking of interacting proteins using self-labeling tags.


Lukinavicius,  G.
Laboratory of Chromatin Labeling and Imaging, Max Planck Institute for Biophysical Chemistry, Max Planck Society;

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Gautier, A., Nakata, E., Lukinavicius, G., Tan, K. T., & Johnsson, K. (2009). Selective cross-linking of interacting proteins using self-labeling tags. Journal of the American Chemical Society, 131(49), 17954-17962. doi:10.1021/ja907818q.

Cite as: https://hdl.handle.net/21.11116/0000-0001-DFDA-F
We have designed molecules that permit the selective cross-linking (S-CROSS) of interacting proteins in cell lysates and the sensitive detection of the trapped complexes through in-gel fluorescence scanning. S-CROSS requires the expression of the putative interacting proteins as fusion to CLIP-tag or SNAP-tag, two protein tags that can be specifically labeled with synthetic probes. Bifunctional molecules that contain the substrates of the two tags connected via a fluorophore are used to selectively cross-link interacting proteins in cell lysate. The amount of trapped complex can be then quantified after SDS gel electrophoresis by in-gel fluorescence scanning. On the basis of a detailed kinetic analysis of the cross-linking reaction, we showed that the cross-linking efficiency can be used as an indicator of interaction between two proteins, allowing thereby the unambiguous identification of interacting protein pairs. We validated our approach by confirming a number of interactions through selective cross-linking and showed that it permits the quantitative and simultaneous analysis of multiple homotypic and heterotypic protein complexes and the differentiation between strong and weak protein−protein interactions.