Fluorescence quenching and ligand binding: A critical discussion of a popular methodology

In recent years fluorescence quenching has become a popular tool to investigate various aspects of ligand binding. Unfortunately, various pitfalls are often overlooked in a large number of papers, published in many different journals. In this criticism we discuss a number of possible mistakes and show how they may affect the data and their analysis. Moreover, we point to problems in the understanding of the fundamentals of fluorescence quenching, and show direct contradictions within many of these papers. This review hopefully contributes to a re-appraisal of the published literature and to a more appropriate use of fluorescence quenching to study ligand binding.

► Ligand binding analysis by fluorescence quenching methodology is often performed incorrectly.
► Typical pitfalls of the methodology and potential impacts are discussed.
► Proper methodology is briefly indicated.
► Many articles will require re-evaluation.