Protocol for evaluating protein-polyfluoroalkyl substances in vitro using differential scanning fluorimetry

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous synthetic chemicals that threaten public health, and serum albumin binding of PFAS represents one major variable influencing PFAS toxicokinetics. In this protocol, we describe a differential scanning fluorimetry (DSF) assay suitable for the rapid determination of the relative binding affinities of serum albumin proteins to different PFAS. Herein, we address common experimental challenges related to PFAS solubility constraints, the high background fluorescence of DSF with serum albumins, and the limitations of using DSF-derived dissociation constants (KD) to quantify PFAS-albumin interactions. For complete details on the use and execution of this protocol, please refer to Jackson et al.1 [Display omitted] •Protocol for the rapid determination of protein-PFAS relative binding affinities•Instructions for the safe preparation of PFAS chemical stocks•Guidance for handling PFAS solubility constraints•Guidance in the interpretation and utility of DSF KD values Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Per- and polyfluoroalkyl substances (PFAS) are ubiquitous synthetic chemicals that threaten public health, and serum albumin binding of PFAS represents one major variable influencing PFAS toxicokinetics. In this protocol, we describe a differential scanning fluorimetry (DSF) assay suitable for the rapid determination of the relative binding affinities of serum albumin proteins to different PFAS. Herein, we address common experimental challenges related to PFAS solubility constraints, the high background fluorescence of DSF with serum albumins, and the limitations of using DSF-derived dissociation constants (KD) to quantify PFAS-albumin interactions.