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

基于亲和力的抗体特异性，用于表征体外和体内神经末梢痛觉过敏信号中的 PKA 亚型

• 批准号: 459759629
• 负责人: Professor Dr. Friedrich-Wilhelm Herberg
• 金额: --
• 依托单位: Klinik für Anästhesiologie und Operative Intensivmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/459759629?language=en
• 关键词: Affinity; based; antibody; specification; characterization

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.

由于背景结合和交叉反应性的范围大多未知，抗体是蛋白质定位的不可靠工具。目前用于抗体应用技术优化的方案很容易出现伪影。我们的目标是开发一种通用的计算工具，以基于简单的测量来识别结合位点的数量以及亲和力，这可以在任何实验室中使用例如免疫荧光显微镜。我们将通过两个应用示例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 亚型产生的最佳纳米抗体，以检测其各自调节亚基的活性依赖性构象。其次，我们将使用这些纳米抗体首次分析体外培养的神经元中伤害性神经元的神经突以及体内完整角膜组织的神经末端中同种型特异性 PKA 活性。