Department of Biochemistry, Stanford University, Stanford, United States.
Department of Chemical Engineering, Stanford University, Stanford, United States.
Elife. 2020 Aug 6;9:e57264. doi: 10.7554/eLife.57264.
Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models. Given the advances in high-throughput technologies and the projected increase in the availability of binding data, we found it especially timely to evaluate the current standards for performing and reporting binding measurements. A review of 100 studies revealed that in most cases essential controls for establishing the appropriate incubation time and concentration regime were not documented, making it impossible to determine measurement reliability. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. Given these challenges, we provide a framework for a broad range of researchers to evaluate, teach about, perform, and clearly document high-quality equilibrium binding measurements. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4.
生物分子相互作用的定量测量是生物学理解的核心，并且需要构建和测试预测和机制模型。鉴于高通量技术的进步以及预计绑定数据可用性的增加，我们发现特别及时评估当前进行和报告绑定测量的标准。对 100 项研究的回顾表明，在大多数情况下，未记录建立适当孵育时间和浓度范围的基本对照，使得无法确定测量的可靠性。此外，可以得出结论，一些报告的亲和力是不正确的，从而影响生物学解释。鉴于这些挑战，我们为广泛的研究人员提供了一个框架，以评估、教授、进行和清晰记录高质量的平衡结合测量。我们通过具有 RNA 结合蛋白 Puf4 的实验示例应用此框架并解释基本概念。
