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

抗体是不可靠的蛋白质定位工具，因为大多数未知的背景结合和交叉反应的延伸。目前抗体应用的技术优化方案容易产生伪影。我们的目标是开发一种通用的计算工具，以确定结合位点的数量以及基于简单测量的亲和力，这可以在任何实验室使用免疫荧光显微镜进行。我们将在两个应用程序示例中证明这种技术的重要性。首先，我们将使用这些工具来选择针对蛋白激酶A同种异构体提出的最佳纳米体，以检测其各自调节亚基的活性依赖构象。其次，我们将使用这些纳米体首次分析体外培养神经元中痛感神经元的神经突以及体内完整角膜组织中的神经末梢中同种型特异性PKA活性。

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.