Abstract
Analysis of cellular signal transduction processes increasingly focuses on the systematic characterization of complete protein interaction networks. Understanding the interplay of signaling components enables insight into the molecular basis of diverse diseases such as cancer. This paves the way for the rational design of specific therapeutics. Protein interactions are often mediated by conserved ...
Abstract
Analysis of cellular signal transduction processes increasingly focuses on the systematic characterization of complete protein interaction networks. Understanding the interplay of signaling components enables insight into the molecular basis of diverse diseases such as cancer. This paves the way for the rational design of specific therapeutics. Protein interactions are often mediated by conserved modular domains, e.g., SH3-domains, which recognize proline-rich sequences in their cognate ligands. In the course of this study, different microarray formats (reactive silane monolayers and nitrocellulose on glass slides) and assay work flows were evaluated to develop a microarray based screening assay that permits the reliable identification of interactions between certain target proteins with a set of SH3 domains. Nine representative SH3 domains which were produced and purified as GST-fusion proteins were spotted on the microarray substrates and probed with two well-characterized ligands, the Nef protein from HIV-1 and the human protein Sam68. The best results from these low-density model arrays were obtained with nitrocellulose slides. We show that a straightforward and highly robust detection of ligand binding is achieved by staining with a fluorescently labeled antibody directed against the N-terminal His-tag attached to these proteins. The optimized assay protocol reported here allows for the identification of SH3-interactions with high reproducibility and adequate signal-to-background and signal-to-noise ratios, as well as the quantitative determination of relative binding affinities.
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