Affinity-based antibody-specification for characterization of PKA-isoforms in hyperalgesic signaling in nerve terminals in vitro and in vivo

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Antibodies are unreliable tools for protein localization due to mostly unknown extend of background binding and crossreactivity. Current protocols for technical optimization of antibody abplications are prone to artefacts. We aim to develop a computational tool for general use to identify the number of binding sites as well as the affinity based on simple measurements, which can be performed in any laboratory using e.g. immunofluorescence microscopy. We will proof the importance of such a technique on two application examples. First we will use the tools to select the best nanobodies raised against isoforms of the protein kinase A to detect activity dependent conformations of their respective regulatory subunits. Second, we will use those nanobodies to analyze for the first time isoform-specific PKA activity in neurites of nociceptive neurons in cultured neurons in vitro as well as in the nerve termini in the intact tissue of the cornea in vivo.