AF4-to-SAXS: expanded characterization of nanoparticles and proteins at the P12 BioSAXS beamline.

Biological small-angle X-ray scattering (SAXS) is a versatile and powerful technique for investigating the structural and biophysical properties of biologically and pharmaceutically relevant macromolecules and nanoparticles. SAXS offers detailed insights into macromolecular composition, size, shape and internal structure, while addressing key aspects such as oligomeric state, stability, molecular interactions, and conformational flexibility. Recently, asymmetrical-flow field-flow fractionation (AF4) was successfully coupled to SAXS, enabling online size-based fractionation and analysis of polydisperse samples. This approach allows precise, size-dependent characterization, offering significant advancements in the study of polydisperse systems. We have integrated an AF4 device at the P12 beamline at the European Molecular Biology Laboratory and implemented technical adaptations allowing full automation to make the system suitable for routine user access. We provide streamlined workflows and troubleshooting resources for both novice and advanced SAXS users thereby equipping them with clear guidance on performing AF4-SAXS measurements. The general principles of our set-up are easily adaptable to other beamlines which have integrated (or are planning to integrate) a similar system.