We apply these procedures with experimental examples and also demonstrate the pitfalls of omitting essential controls. We focus on a minimal set of critical actionable steps and controls that biologists of any background should be able to implement in their binding measurements. The principles underlying these procedures have been discussed and we build on these previous reports ( Pollard, 2010 Hulme and Trevethick, 2010 Sanders, 2010). Fortunately, there are straightforward procedures, laid out here, that can be followed to ensure that published binding measurements are reliable. Our survey of 100 literature binding measurements, presented below, uncovered recurring problems with a large majority of studies. We wanted to know whether current practices are sufficient to ensure reliable and accurate measurements, and whether the reliability of these measurements can be readily ascertained from the information provided in published work. Given these potentially transformative advances, it is especially timely to assess the accuracy of equilibrium binding measurements. Buenrostro et al., 2014 Tome et al., 2014 Lambert et al., 2014 Nutiu et al., 2011 Maerkl and Quake, 2007 Adams et al., 2016 Jain et al., 2017). Excitingly, several strategies have recently emerged to obtain high-throughput, quantitative information for intermolecular associations (e.g. Thus, equilibrium constants for association between network components are needed to define, model, predict, and ultimately precisely manipulate biology.Ī limitation of traditional biochemical measurements is their low throughput, especially in relation to the large number of cellular interactions. The outputs of pathways and networks are determined by the quantitative interplay of their many constituent molecules and interactions.
In a broader biological context, these associations are linked and interconnected in complex networks that allow sensitive and precise developmental programs and responses to environmental cues, and that are altered in disease states. Their thermodynamics provides information critical for deriving a fundamental understanding of molecular functions. Molecular associations lie at the heart of biology. We apply this framework and explain underlying fundamental concepts through experimental examples with the RNA-binding protein Puf4. 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. Moreover, several reported affinities could be concluded to be incorrect, thereby impacting biological interpretations. 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. 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. Quantitative measurements of biomolecule associations are central to biological understanding and are needed to build and test predictive and mechanistic models.