期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 131, 期 49, 页码 17954-17962出版社
AMER CHEMICAL SOC
DOI: 10.1021/ja907818q
关键词
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资金
- European Research Training Network PROSA
- JSPS
- FEBS
- Swiss National Science Foundation
- Grants-in-Aid for Scientific Research [21710232] Funding Source: KAKEN
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.
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