Bates Timothy A, Gurmessa Sintayehu K, Weinstein Jules B, Trank-Greene Mila, Wrynla Xammy Huu, Anastas Aidan, Anley Teketay Wassie, Hinchliff Audrey, Shinde Ujwal, Burke John E, Tafesse Fikadu G
Department of Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, USA.
Department of Chemical Physiology and Biochemistry, Oregon Health & Science University, Portland, OR, USA.
Nat Protoc. 2025 Apr;20(4):861-883. doi: 10.1038/s41596-024-01079-8. Epub 2024 Nov 21.
Protein-protein interactions underpin nearly all biological processes, and understanding the molecular mechanisms that govern these interactions is crucial for the progress of biomedical sciences. The emergence of artificial intelligence-driven computational tools can help reshape the methods of structural biology; however, model data often require empirical validation. The large scale of predictive modeling data will therefore benefit from optimized methodologies for the high-throughput biochemical characterization of protein-protein interactions. Biolayer interferometry is one of very few approaches that can determine the rate of biomolecular interactions, called kinetics, and, of the commonly available kinetic measurement techniques, it is the most suitable for high-throughput experimental designs. Here we provide step-by-step instructions on how to perform kinetics experiments using biolayer interferometry. We further describe the basis and execution of competition and epitope binning experiments, which are particularly useful for antibody and nanobody screening applications. The procedure requires 3 h to complete and is suitable for users with minimal experience with biochemical techniques.
蛋白质-蛋白质相互作用几乎支撑着所有生物过程，理解调控这些相互作用的分子机制对于生物医学科学的进展至关重要。人工智能驱动的计算工具的出现有助于重塑结构生物学方法；然而，模型数据通常需要经验验证。因此，大规模的预测建模数据将受益于用于蛋白质-蛋白质相互作用高通量生化表征的优化方法。生物层干涉术是极少数能够确定生物分子相互作用速率（即动力学）的方法之一，并且在常用的动力学测量技术中，它最适合高通量实验设计。在这里，我们提供了关于如何使用生物层干涉术进行动力学实验的详细分步说明。我们还进一步描述了竞争和表位分组实验的基础及实施，这些实验对于抗体和纳米抗体筛选应用特别有用。该实验步骤需要3小时完成，适合对生化技术经验最少的用户。
