Small-angle X-ray scattering experiments of monodisperse intrinsically disordered protein samples close to the solubility limit.

The condensation of biomolecules into biomolecular condensates via liquid-liquid phase separation (LLPS) is a ubiquitous mechanism that drives cellular organization. To enable these functions, biomolecules have evolved to drive LLPS and facilitate partitioning into biomolecular condensates. Determining the molecular features of proteins that encode LLPS will provide critical insights into a plethora of biological processes. Problematically, probing biomolecular dense phases directly is often technologically difficult or impossible. By capitalizing on the symmetry between the conformational behavior of biomolecules in dilute solution and dense phases, it is possible to infer details critical to phase separation by precise measurements of the dilute phase thus circumventing complicated characterization of dense phases. The symmetry between dilute and dense phases is found in the size and shape of the conformational ensemble of a biomolecule-parameters that small-angle X-ray scattering (SAXS) is ideally suited to probe. Recent technological advances have made it possible to accurately characterize samples of intrinsically disordered protein regions at low enough concentration to avoid interference from intermolecular attraction, oligomerization or aggregation, all of which were previously roadblocks to characterizing self-assembling proteins. Herein, we describe the pitfalls inherent to measuring such samples, the experimental details required for circumventing these issues and analysis methods that place the results of SAXS measurements into the theoretical framework of LLPS.