How to measure and evaluate binding affinities

Abstract
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Abstract

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.

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No datasets were generated in this work. The figures include all data, or, where most appropriate for clarity, representative data from a single experiment for every type of experiment performed.

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Inga Jarmoskaite

Department of Biochemistry, Stanford University, Stanford, United States

For correspondence
ijarmosk@stanford.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-5847-5867
Ishraq AlSadhan

Department of Biochemistry, Stanford University, Stanford, United States

Competing interests
The authors declare that no competing interests exist.
Pavanapuresan P Vaidyanathan

Department of Biochemistry, Stanford University, Stanford, United States

Competing interests
The authors declare that no competing interests exist.
Daniel Herschlag

Department of Biochemistry, Stanford University, Stanford, United States

For correspondence
herschla@stanford.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-4685-1973

Funding

National Institutes of Health (R01 GM132899)

Daniel Herschlag

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.