|WP1: High throughput selection procedures for recombinant affinity binders
* target production for binder selection: clones (SBS), full-length proteins (UU, BAFFIN), peptides (DKFZ)
* optimization of ribosome display (UZH, BBT) and phage display (TUBS)
* selection of antibody fragments (TUBS, BBT), DARPins (UZH), and ssDNA aptamers (SOMA)
* LIMS for data management (TUBS, BBT)
|WP2: Quality control and characterization of affinity binders
Binders generated in WP1 will be ranked for further applications and the selection of pairs for sandwich assays and proximity ligation.
* determination of affinity and binding kinetics (BAFFIN)
* target specificity and cross-reactivity on protein arrays (BBT, DKFZ)
* epitope specificity on peptide arrays (DKFZ)
|WP3: Development and refinement of advanced affinity-based technologies for proteomics
Novel affinity-based applications will be optimized and adapted for high-throughput studies using the binders selected and characterized in WPs 1 and 2.
* capture arrays of immobilized binders for protein expression profiling in extracts (DKFZ, BBT, UNIKASSEL)
* in situ proximity ligation to demonstrate individual proteins and complexes in fixed cells and tissues (OLINK)
* functional intracellular expression in living cells as intrabodies (UZH, UNIKASSEL, TUBS)
|WP4: Applications of novel affinity-based technologies in functional proteomics of signal transduction pathways
Cell signal transduction pathways will be analyzed using the binders and assays developed in WP1-3, with the aim of systematic analysis in a rapid and parallel fashion. The focus will be on interactions from receptors to transcription factors in the TGF-beta and MAPK pathways.
* quantitate expression levels and activation state of the kinases and other pathway proteins by binder arrays (DKFZ, UNIKASSEL, UU)
* determine their cellular localization and physical interactions by Proximity ligation (OLINK).
* examine functional disruption of signaling in living cells with intrabodies (UZH, UNIKASSEL)