ProteomeBinders

ProteomeBinders is strongly considered to be a project which assembles world class expertise in binder technologies, tools and applications for the long-term understanding of the human proteome. It is intended that the integration of resources into the infrastructure will be lasting and continue in a sustainable practical format after the end of the coordination phase. The CA will serve as a springboard for thorough preparation of all aspects of proteome ligand binder technology which lead to the establishment of a full, productive European binder resource over the following years. The benefit of the CA will be to provide a period of coordinated and detailed planning, benchmarking, assessment and integration, such that the practical organisation and productivity of the resource can be put into place efficiently when the (not insignificant) funds are approved.
The Herberg group specialises in real-time analysis of biomolecular interactions employing biophysical techniques, incl. surface plasmon resonance, using Biacore 2000 and 3000 systems. In this CA they will undertake Biomolecular Interaction Analysis for high resolution kinetic analysis of binders and applications in validation and quality control (NA2.6, Binder characterisation).
A major prerequisite for binder analysis on solid supports, e.g. for epitope mapping, is the preservation of functionality upon immobilisation. For this purpose, protein purification strategies are adapted based on 6xHis-, GST-, FLAG- or proprietary dual-affinity-tags for site-directed immobilisation. For increased throughput, bead based technologies like AlphaScreen, a non-radioactive proximity assay (up to 1536-well format) and a variety of methods for fluorescence based or label-free interaction assays (Fluorescence Polarisation; Isothermic Titration Calorimetry) are established. In vitro methods for analysis of protein-protein interactions are accompanied by in-cell assays, i.e. Bioluminescence Resonance Energy Transfer (BRET), allowing validation and quantitation of interactions in living cells in a 96-well microtiter plate format with a multi-label reader. This will be applicable in NA4.2 (Functional analysis of binders, including intrabodies).
The methods have potential for automation, are applicable in medium to high throughput, and are applied in functional analysis of protein kinase networks and scaffolds.

back