|
WP1: High throughput selection procedures for recombinant affinity binders * target production for binder selection: clones (SBS), full length proteins (UU, BIAFFIN), peptides (DKFZ) * optimisation 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 characterisation of affinity binders Binders generated in WP1 will be ranked for further applications and selection of pairs for sandwich assays and proximity ligation. * determination of affinity and binding kinetics (BIAFFIN) * 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 optimised and adapted for high-througput studies using the binders selected and characterised in WPs 1 and 2. * capture arrays of immobilised binders for protein expression profiling in extracts (DKFZ, BBT, UNIKASSEL) * multidimensional fluorescence microscopy for protein colocalisation studies (REVOTAR) * 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 analysed 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 factorsin 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 localisation and physical interactions by Multidimensional Fluorescence Microscopy (REVOTAR) and Proximity ligation (OLINK). * examine functional disruption of signalling in living cells with intrabodies (UZH, UNIKASSEL) |